Yaru Xie,Lei Chen,Tao Sun,Weiwen Zhang,4,5,*

1 Laboratory of Synthetic Microbiology,School of Chemical Engineering &Technology,Tianjin University,Tianjin 300072,China

2 Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering,Ministry of Education of China,Tianjin 300072,China

3 Collaborative Innovation Center of Chemical Science and Engineering,Tianjin 300072,China

4 Center for Biosafety Research and Strategy,Tianjin University,Tianjin 300072,China

5 Law School,Tianjin University,Tianjin 300072,China

ABSTRACT Development and utilization of“liquid sunshine”could be one of key solutions to deal with the issues of fossil fuel depletion and increasing carbon dioxide.Cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis,and their activity accounts for～25% of the total carbon fixation on earth.More importantly,besides their traditional roles as primary producers,cyanobacteria could be modified as “photosynthetic cell factories”to produce renewable fuels and chemicals directly from CO2driven by solar energy,with the aid of cutting-edging synthetic biology technology.Towards their large-scale biotechnological application in the future,many challenges still need to be properly addressed,among which is cyanobacterial cell factories inevitably suffer from high light(HL)stress during large-scale outdoor cultivation,resulting in photodamage and even cell death,limiting their productivity.In this review,we critically summarized recent progress on deciphering molecular mechanisms to HL and developing HL-tolerant chassis in cyanobacteria,aiming at facilitating construction of HLresistant chassis and promote the future application of the large-scale outdoor cultivation of cyanobacterial cell factories.Finally,the future directions on cyanobacterial chassis engineering were discussed.

Keywords:High-light Stress Chassis Cell factories Cyanobacteria

1.Introduction

Fossil fuels are non-renewable resources and their increasing utilization releases significant amount of greenhouse gases like carbon dioxide into the atmosphere,which has been considered as one of the main causes of global warming.It is therefore an urgent challenge to explore renewable,green and clean energy to replace traditional fossil energy and alleviate environmental pollution.Via photosynthesis,about 258 billion tons of CO2are fixed into organic matter on earth each year,of which～70% is contributed by marine algae and photosynthetic bacteria.Among them,cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis,and their activity accounts for～25%of the total carbon fixation on earth [1,2].

Besides their traditional roles as primary producers,with the aid of cutting-edging synthetic biology technology in recent decades,cyanobacteria have been engineered as“light-driven cell factories”to produce dozens of renewable fuels and chemicals including ethanol,butanol,butanediol,propanediol,acetone,sucrose,limonene and 3-hydroxypropionic acid directly from CO2[3].Notably,recent development and application of synthetic biology and multiple metabolic engineering tools enabled modification of model cyanobacteria likeSynechocystissp.PCC 6803 (hereafterSynechocystis) andSynechococcus elongatusPCC 7942 to produce biochemicals with a high productivity [3–5].For example,introduction of pyruvate decarboxylase fromZymomonas mobilisand overexpression of endogenous alcohol dehydrogenase inSynechocystissuccessfully reached ethanol production of 5.5 g·L-1[6].In addition,by introducing several heterogenous genes as well as modifying the native glycolytic pathways and the carbon fixation,the engineeredS.elongatusPCC 7942 efficiently used both CO2and glucose,synthesizing 12.6 g·L-12,3-butanediol under continuous light conditions [7].Moreover,overexpression of the sucrose synthesis genes and sucrose transporter encoding genecscBinS.elongatusUTEX 2973 obtained 8 g·L-1of sucrose under salt stress condition[8].All these studies exclusively demonstrated the feasibility of developing sustainable production systems based on photosynthetic cyanobacterial cells.

Industrial or large-scale cultivation of cyanobacteria have to depend on outdoor cultivation to utilize natural solar energy to make it cost-effective[9].For example,outdoor cultivation of some cyanobacteria likeArthrospira platensisorAnabaenasp.has been evaluated in 800 L or 2000 L open ponds,reaching a biomass at～1.0 g·L-1[10,11].For outdoor cultivation,it is costly to maintain the consistent sunlight and cyanobacteria would inevitably encounter high light (HL) stress in nature,which could cause severe damage to the cells and greatly reduce their photosynthetic productivity.Therefore,deciphering and engineering high-light tolerant cyanobacteria will promote the construction of efficient photosynthetic cell factories for industry application.

In recent years,combined technologies especially multiple omics and systems biology allowed for a comprehensive survey of the gene expression profile during HL acclimation of cyanobacteria [12,13].In addition,constructions of robust cyanobacterial chassis against HL stress have been successfully achieved to some degree with synthetic biology [14–16].In this review,we briefly introduce the cyanobacterial photosystem and then critically summarize the recent progress on photoprotection mechanisms.In addition,we also discuss the strategies to improve the tolerance of cyanobacteria to HL,and provide our insights on future research direction in the field.

2.High Light Stress on Photosystems

Microbial photosystems typically consist of photosystem I(PSI),photosystem II(PSII)and cytochrome b6f complex(cytb6f)(Fig.1).Among them,PSI is a multi-subunit protein complex which contains 14 subunits (i.e.,PsaA-PsaL,PsaN and PsaO) in the central part of the complex[17]while PSII is a large protein subunit dimer compound,each monomer made up of oxygen-evolving complex(OEC),D1,D2,CP43 and CP47 etc.(for more details,please refer to ref.[18]).In addition,the cytb6f complex contains eight subunits,functioning as a plastoquinol-plastocyanin (cytochrome c)reductase which connects electrons transport between PSII and PSI [19],forming the photosynthetic electron transport chain together with the mobile electron carriers plastoquinone (PQ)and plastocyanin [18].Notably,PSII is the starting point of photosynthetic electron transport chain which catalyzes the reaction of light-driven water oxidize and transfers electron from water molecule to PQ [17].Then,electrons flow through cytb6f,accompanied by generation of the proton electrochemical potential gradient which is necessary for ATP synthesis [20].At last,PSI accepts an electron from plastocyanin and transfers it to its major acceptor,ferredoxin and then turns NADP+into NADPH upon illumination[17,21].The electrons from water transported via PSII,cytb6f and PSI finally generate ATP and NADPH,which are further utilized for the fixation of CO2[22].The major light harvest device of PSII is phycobilisome and a few chlorophyll molecules (Chl),while a large core antenna system consisting of 90 Chla molecules and 22 carotenoids is utilized by PSI [23,24].

Early studies showed that excess light energy can break the balance between energy supply and consumption in cyanobacteria,leading to the intracellular accumulation of reactive oxygen species (ROS) including hydrogen peroxide (H2O2),superoxide (O2–),hydroxyl radicals(OH▪),and singlet oxygen(1O2)[12].The accumulated ROS further disrupts other key cellular components like nucleic acids,lipids,pigments and proteins,leading to cell death[25,26].Detailly,photosynthesis can be considered as a series of sequential redox reactions.Photosynthetic redox balance is maintained when light energy supply is equal to energy consumption but balance will be broken when excess light is harvested [27],leading to the over-reduced PQ pool and electron transport chain[28].In addition,PSII is more vulnerable to HL intensity than PSI,and HL causes photodamage to PSII reaction centers [29].Two hypothesesi.e.,the two-step photodamage model and acceptorand donor-side photoinhibition models,have been proposed in the past to explain the photodamage mechanisms to PSII [30].According to the first model,the initial photodamage of PSII is the water splitting site in the oxygen evolving complex[30],while in the second model,the initial photodamage is reaction center associated with PSII electron transport [31].

Fig.1.Schematic representation of the responding mechanisms of cyanobacteria to HL.PSII:photosystem II;D1:one of reaction center of PSII;PSI:photosystem I;Cytb6f:cytochrome b6f complex;ATP synthetase:Adenosine triphosphate synthetase;PQ:plastoquinone;PC:plastocyanin;Fd:ferredoxin FNR:NADP oxidoreductase;NPQ:nonphotochemical quenching;OCP:orange carotenoid protein,OCPo:the inactive orange form of OCP;OCPr:the active red form of OCP;FRP:fluorescence recovery protein(OCP was mediated via NPQ process under HL;OCPo was converted to OCPr to transfer energy into heat then returning to its original state by FRP);CET:circular electron transfer;LET:linear electron flow;PGR5:the polypeptide proton gradient regulation 5;NDH:plastoquinone reductase;PGR5 and NDH mediated circular electron transfer;Flv:flavodiiron proteins;FtsH and deg:enzyme for degrading damaged D1 protein;SOD:superoxide dismutase;ROS:reactive oxygen species;HCP:the helical carotenoid proteins;2PG/3PG:2-phosphoglycolate/3-phosphoglycolate;RUBP:ribulose-1,5 bisphosphate;RuBisCO:ribulose-1,5-bisphosphate carboxylase-oxygenase;NADPH:nicotinamide adenine dinucleotide phosphate.

3.HL Acclimation Mechanisms in Cyanobacteria

During the long history of evolution,cyanobacteria have developed a series of strategies to survive under HL stress.It is thus expected that deciphering the molecular responses and photoprotection mechanisms of cyanobacteria to HL could provide the most needed guidance to engineer robust and tolerant strains.In general,several strategies,including reduction of light absorption,excess electron and energy utilization,acceleration of turnover of the PSII protein and internal light protection mechanism,are all utilized by cells[12](Fig.1),and recent analysis of these strategies led to the identification of key genes and networks involving in the HL stress responses [12],which are summarized below.

3.1.Reduced harvest of light energy

Similar to plants,cyanobacteria have evolved an excessive lightharvesting antenna,i,e,the phycobilisome(PBS)to guarantee their survival advantage over other species when growing under extremely light-limiting conditions (Fig.1).Vice versa,the photon absorption would far exceed the demands of photosystems when under NL or HL,forcing cells to dissipate redundant energy into heat or other forms.As the major harvest antenna,PBS and Chl would decline their sizes and/or contents to avoid absorbing too much light energy as a way of protecting photosystems under HL[32].Detailly,inSynechocystis,PBS encoding genescpcandapcwere down-regulated,followed by a decrease in the length of PBS rods and PBS content per cell [12,33].Early study also found that,the stability of polycistronic linker protein mRNAs was decreased markedly from 16 min to 1 min whenSynechococcus elongatusPCC 6301 encountered HL condition [34].In addition,PBS could move between PSI and PSII under HL [35,36],redistributing light energy between PSII and PSI to protect PSII from excessive light energy [36].Moreover,as the main antenna of PSI,the synthetic pathway of Chl could be down-regulated under HL.There are six different types of chlorophyll (Chls a,b,d,f and divinyl-chls a and b) naturally present in cyanobacteria,among which chl a was the most distributed chlorophyll pigment among various species [37].Chlorophylls,hemes,and bilin pigments are synthesized via a common tetrapyrrole biosynthetic pathway among photosynthetic organisms.As one of pivotal control points of the pathway,the level of 5-aminolaevulinic acid(ALA)synthesis is involved in regulating the content of Chl under HL[12].Upon the shift to HL conditions,synthesis of ALA was down-regulated [38].In addition,the gene related to Chl synthesis such ashemA(slr1808),ho1(sll1184) andhemF(sll1185) were all downregulated under HL inSynechocystis[12,33],leading to the decreased chlorophyll.

3.2.Quenching excess light energy

To avoid excessive light entering photosystem reaction center,cyanobacteria have evolved nonphotochemical quenching (NPQ)to dissipate energy into heat [39](Fig.1).Orange carotenoid protein (OCP) is a soluble 35 kDa pigment-protein complex which is essential for triggering NPQ-mediated photoprotective mechanism[40].Detailly,the inactive orange form (OCPo) is converted to the active red form (OCPr),and then return to its original state combined with FRP (Fluorescence Recovery Protein) [41].In a recent study,a novel carotenoid carrier of OCP named COCPwas found capable of efficiently transferring carotenoid to the full-length OCP apoprotein [42].In addition,new paralogous families of OCP(OCPX,OCP2) and its domain homologs,the Helical Carotenoid Proteins (HCPs) and C-terminal domain homologs have also been demonstrated distributed widely among cyanobacteria [43,44].Notably,some of them like HCP2 and HCP3 exhibit strong singlet oxygen (1O2) quenching capability,while HCP4 and OCP2 are capable of dissipating PBS excitation [44–48].OCP energy quenching can be rapidly induced and reversed,on a timescale of seconds to minutes.

Besides,three protein complexes that bind with carotenoid have also been identified inSynechocystis,including a novel high light-inducible carotenoid-binding protein complex (HLCC)encoded byslr1128,isiA,psaD,andhliA/B,and functioning as a protector in the thylakoid membranes of theSynechocystiscells when exposed to HL [49],and another complex consisted of HliDchlorophyll-a (Chl-a) and β-carotene exhibits an energydissipative conformation [50].Interestingly,a recent study(2016) found that both complexes are composed of Chl synthase(ChlG),HliC-D binding with a Chl a and three different carotenoids(i.e.,β-carotene,zeaxanthin and myxoxanthophyll) [51].Notably,the two complexes quench superfluous energy via direct energy transfer from a Chl-a Qystate to the b-carotene S1state [50,51].

3.3.Induction of alternative electron transport pathways

Alternative electron transport is utilized by photosynthetic organisms to respond rapidly to changes in their environments(Fig.1).Alternative electron transport pathways in cyanobacteria consist of photorespiration,cyclic electron transport and flavodiiron protein mediated electron transport pathway.Besides the normal reaction with CO2,ribulose 1,5-bisphosphate could also react with O2and generate 2-phosphoglycolate,which is toxic to cyanobacteria metabolism via inhibiting distinct steps in the carbon-fixing Calvin-Benson cycle.Cyanobacteria employ photorespiratory pathway consuming extra ATP and NADPH to convert 2-phosphoglycolate into 3-phosphoglycolate,CO2and NH4+.Although regarded as the high energetic cost and wasting energy pathway,there is no doubt that photorespiration plays an essential role in protecting cells from HL [52].It has been estimated that photorespiration pathway can consume up to 43%–55%of the total electron flow through photosystem II under HL [52].

Cyanobacteria typically exhibit two modes of cyclic electron transport (CET) around PSI,FQR cycle and NDH-1 cycle,in which electrons are returned from the acceptor side of PSI back to the donor side of PSI via the photosynthetic electron transport chain.CET is particularly important for controlling the redox balance for cells under environmental stress conditions such as low CO2,high light,drought or dark to light transitions[53].Under fluctuating light,CET could prevent damage to PSI by providing a provisional electron sink downstream of PSI [54].Cyclic electron flow contributes toward production of the “extra”ATP that is crucial for the proper balance of NADPH and ATP and the protection of PSII from photoinhibition by providing ATP for repair of PSII units damaged by environmental stress [55,56].

Flavodiiron (Flv) proteins are involved in O2photoreduction by transferring electrons from PSI and PSII to O2without the release of ROS[57].In cyanobacteria,Flv proteins function as a large electron sink downstream of PSI and PSII,with >(20–30)% of electrons derived from PSI and PSII participating in O2photoreductionviaFlv1/Flv3 or Flv4/2 heterodimer under HL,respectively [57,58].In one study,theflv2andflv4double deletion mutant was found to be susceptible to high light intensities under ambient CO2[59].In another study,the growth of a knockout mutant of Flv became arrested in the most severe and long-term fluctuating light conditions,ultimately resulting in cell death[60].It is concluded that Flv proteins are important for survival of cyanobacteria under NL or HL due to their roles in forming a large electron sink [60].Flv2 and Flv4 proteins are limited to β-cyanobacteria and function in photoprotection of PSII,while Flv1 and Flv3 proteins occur in nearly all cyanobacteria,protecting PSI under fluctuating light conditions.Notably,overexpression of theflv4/2operon inSynechocystisresulted in improved photochemistry of PSII and the resistance of cells to HL [58].Alternative electron transport can transfer redundant electron into ATP or water without releasing ROS which can be an ideal target to enhance HL tolerance of cyanobacteria.

3.4.Acceleration of turnover of the PSII protein and removal of ROS

The D1 protein forming a heterodimer with the D2 protein in the reaction center of PSII is the major target of photodamage in PSII (Fig.1).To deal with photoinhibition,photosynthetic organisms have evolved a PSII repair cycle to rescue the photodamaged PSII reaction center.Many cyanobacteria contain additional copies of thepsbAgene encoding the D1 protein,whose expression is induced by HL under regulation by Hik33.For example,S.elongatusPCC 7942 possesses two isoforms of D1 protein:the D1:1 form encoded bypsbA1and the photodamage-resistant D1:2 forms encoded bypsbA2andpsbA3[61].Expression of thepsbA2andpsbA3genes is inducible by HL,whilepsbA1gene is actively transcribed under normal condition.InSynechocystis,three copies ofpsbAgenes:psbA1,psbA2,andpsbA3have been identified.Upon a shift to HL condition,bothpsbA2andpsbA3were up-regulated inSynechocystis[12].On the other hand,the damaged D1 protein must be rapidly degraded and removed in order to be replaced by the newly synthesized or repaired D1 protein [62,63].Consistently,theftsHanddeg/htrfamily related with D1 degradation were found up-regulated under HL condition [64–66].

It has been demonstrated that ROS could cause photoinhibition through inhibition of PSII repair.Cyanobacteria contain robust antioxidant and redox buffering systems composed of enzymatic and small molecule components,such as peroxidases,glutathione reductase,ascorbate,glutathione as well as carotenoids and tocopherols to scavenge excess ROS [67–69].One of the most ubiquitous protective enzymes,superoxide dismutase (SOD) have been found in many cyanobacteria,such asSpirulina platensis,Pseudanabeanasp.andSynechococcus nidulans[70].Most of cyanobacteria are known to contain both FeSOD and MnSOD [70],while Cu/ZnSOD and NiSOD were mainly found in filamentous nitrogenfixing cyanobacteria such asAnabaena variabilisATCC 29413 andNostocPCC 7120 [71].Those related pathways serve as potential targets for engineering HL-tolerant cyanobacterial chassis.

4.Strategies to Improve HL Tolerance in Cyanobacteria

As discussed above,combined with multiple omics technology and systems biology,series of target genes related with HL stress response have been gradually identified in recent decades.Driven by synthetic biology,various strategies to enhance the tolerance to HL were also applied (Table 1).

4.1.Reduction of antenna sizes

In early studies,reduction of antenna size has been demonstrated to enhance not only the tolerance to HL but also cyanobacterial biomass and productivity.For example,Kirstet al.(2014)achieved a phycocyanin-deletion mutant (Δcpc) ofSynechocystiswith a lower Chl per cell content and a lower PSI/PSII reaction center ratio(wild type(WT):2.5;Δcpc:1.8),and the mutant exhibited2 times saturation of photosynthesis higher than WT [72].In addition,culture productivity analysis showed that biomass accumulation by the Δcpcwas 1.57-times greater than that achieved by the WT under HL condition(2000 μmol photons·m-2-·s-1) [72].In another study,Nagarajanet al.(2014) exploited the phenotypes of three mutants with deleted phycobilisomes variants CB (phycobilisomes rods containing only one phycocyanin hexamer),the CK (containing only the allophycocyanin core),and the PAL (cannot assemble any functional phycobilisomes) [73,74].The results showed that the PAL mutant showed a 50% decrease in PSI when compared to WT while CK and PAL mutants showed maximum rates of oxygen evolution under extreme light condition(up to 8000 μmol photons·m-2·s-1)with～4 folds higher than those measured for WT [73].Josephet al.(2014) deletedapcEgene encoding the anchor protein linking the phycobilisome to the thylakoid membrane,and obtained a ΔapcEmutant that grew much better than WT under 200 μmol photons·m-2·s-1[75].After 15 days’ cultivation,the ΔapcEmutant accumulated 1.6 times more biomass and glycogen content than WT under HL.In addition,the ΔapcEmutant showed higher oxygen evolution rate at 600–1800 μmol photons·m-2·s-1,suggesting that the lightsaturated evolution (Pmax) was higher in the ΔapcEmutant.Consistently,similar results were also found by knocking out PC hexamers [76].All these studies clearly demonstrated that reduction of antenna size can be feasible strategies to enhance the cyanobacterial tolerance and biomass accumulation under HL.However,most of the current studies on reduced antenna focused only on knockout of the key genes related with phycobilisome.In addition,sunlight intensity is varying during a day,thus the direct reduction of antenna may affect the sunlight utilization and cyanobacterial growth due to the insufficient photon absorption during the dim time.To address this issue,more timely and dynamic regulation of the antenna size in response to the changing light intensity through the day should be a new focus in the future.

Table 1 Major strategies to enhance tolerance of cyanobacteria to HL.(The unit for light intensity was μmol photons·m-2·s-1)

4.2.Enhancement of photoprotective pathways

Early studies have showed that overexpression of photoprotection related genes could enhance its HL tolerance in plants[86,87].In cyanobacteria,OCP mediated NPQ process has been considered as the main photoprotective pathway under HL [40,88].Interestingly,overexpression of OCP (ox-ocp) inSynechocystisnot only increased thermal dissipation of energy but also alleviated PSII photoinhibition degree by protecting repair pathway of PSII under HL [14].Detailly,the loss of PSII activity of ox-ocp was 2 times slower than WT when exposed to HL at 1,500 μmol photons·m-2·s-1,indicating that PSII in ox-ocp cells was more resistant to photoinhibition [14].In addition,the rate of D1 protein synthesis in ox-ocp cells was 1.5 times higher than that in WT,suggesting that overexpression of OCP might accelerate the synthesis of the D1 protein under HL as one mean of protecting PSII.Moreover,overexpression of an iron superoxide dismutase(Fe-SOD)and VktA inS.elongatusPCC 7942 increased HL tolerance by scavenging ROS[15].Furthermore,overexpressing EF-Tu inSynechocystisachieved HL tolerance up to 1,500 μmol photons·m-2·s-1,probably due to the fact that EF-Tu accelerated PSII repair rate [79].In theory,Fe-SOD,VktA and EF-Tu can be combinedly utilized as targets to further improve the HL tolerance in the future.

Some genes are known to regulate protective pathways negatively thus their knocking out could be used as a way of activating photoprotective pathways.For example,Mirandaet al.(2017)reported that deletion ofsll1783related to polysaccharide degradation improved the growth by 1.7 fold than WT under HL inSynechocystis[80],although the role of Sll1783 against HL stress is still unclear.In addition,Group 3 sigma factors are thought to be important for cyanobacteria to adapt environmental challenges by altering expression of the genes necessary for coping with such stresses [80].Srivastavaet al.(2016) found that knocking down of thesigJgene increased tolerance ofAnabaenasp.PCC 7120 to HL through an increased accumulation of myxoxanthophyll and keto-myxoxanthophyll in cells [81].Moreover,Khanet al.(2016)found that a transcriptional regulator,PrqR negatively regulated glucose metabolism and ROS removal pathway by controlling the expression ofsodBandprqA(slr0896) [89].TheprqRmutant was found with increased growth rate and decreased ROS content under HL [89].These studies independently demonstrated the roles of key genes in enhancing the tolerance to HL.In the future,co-overexpression of all or selected group of these genes should be carried out to verify whether superimposed effects can be achieved.Meanwhile,additional metabolic burden caused by the overexpression of too many genes simultaneously should also be properly addressed.

4.3.Engineering alternative electron and carbon sinks

Under HL,excess electron or energy produced by photosystem cannot be timely consumed by the downstream carbon fixation process,leading to more ROS formed.Therefore,HL tolerance of cyanobacteria could also be achieved through constructing alternative electron and carbon sinks.For example,flavodiiron protein was able to alleviate PSII excitation pressure up to 30% [16,58],theSynechocystisoverexpressed withflv4/flv2(flv4/2/OE) grew approximately 3 times faster than the WT and remained green phenotype even after 7 days’ cultivation under 1,500 μmol photons·m-2·s-1[58].In addition,theflv4/2/OE mutant presented significantly higher PSII activity and lower1O2content,as evidenced by～10% higher oxygen evolution rates compared with the WT under HL [58].These results indicated that overexpression offlv4/2could protect PSII from photoinhibition and improve photochemistry of PSII by dissipating excitation pressure of PSII.Overexpression offlv3inSynechocystisalso exhibited a protective role under HL.Under 120 μmol photons·m-2·s-1light intensity,biomass and glycogen content of theflv3overexpression strain were 29% and 28% higher than those of WT,respectively [16].Besides,heterologous electron sinks could also be utilized to improve HL tolerance in cyanobacteria.For example,Berepikiet al.(2016)expressed the mammalian cytochrome P450 CYP1A1 as an artificial electron sink for excess electrons derived from lightcatalyzed water-splitting inSynechococcussp.PCC 7002,resulting in an increased maximum rate of photosynthetic electron flow by～31.3% [83].

The low effective quantum yield of natural light-harvesting systems under HL was partially caused by downstream limitations[90].The down-regulation of photosynthesis caused by reduced capacity to utilize photosynthate has been termed as “sink limitation”[91].Sink limitation can lead to an accumulation of soluble sugars,depletion of intracellular free phosphate,lowered ATP synthase activity and elevated proton motive force [91].Triggering high levels of exporting photosynthetically produced energy carriers from cyanobacteria may be a feasible and attractive approach to enhance the rate and effective photon yield under HL [90].As typically carbon sinks in cyanobacteria,sucrose and glycogen biosynthesis could be engineered to improve HL tolerance by assuming a large portion of carbon and energy from the CBB cycle[92].Besides,heterologous pathway for enhancing the production of natural or non-natural metabolites could also be introduced as an extra carbon sink to partially replace the functions of the natural one[92].In an early study,a symporter of protons and sucrose(cscB) was heterologously expressed inS.elongatusPCC 7942 to increase not only sucrose yield but also photosynthetic efficiency[93].Detailly,sucrose-exporting cyanobacteria exhibited an increased biomass production relative to WT,accompanied by enhanced photosystem II activity (～20%),carbon fixation (20%),and chlorophyll content (～25%) [93].In another study,Abramsonet al.demonstrated that the magnitude of the increase in PSII operating efficiency was correlated with the rate of sucrose export[91].Furthermore,developing the 2,3-butanediol biosynthetic pathway inS.elongatusPCC 7942 displayed a slightly higher rate of O2evolution per microgram of chlorophyll during late stages of production compared with control strains [94].Similarly,another study demonstrated that isobutanol can also be used as a carbon sink which was able to partially rescue the growth of strains lacking glycogen synthesis ability [95].Given that cyanobacterial chassis have been engineered to synthesize multiple compounds including biofuels,biochemicals and nutriments [3–5],utilizing these compounds as extra carbon sinks to fight against HL might be a win–win strategy.In the future,more native and non-native products or pathways should be evaluated for their efficiency as carbon sinks in cyanobacteria.

4.4.Introducing key genes from HL-tolerant cyanobacterium

Several new cyanobacterial species includingS.elongatusUTEX 2973,S.elongatusPCC 11801,S.elongatusPCC 11802 andSynechococcussp.PCC 11901 have been recently identified,with great application properties like fast growth and great tolerance to HL,high temperature,high NaCl and high CO2[96–99].Interestingly,the genome homology of the four strains were found highly identical respectively to model cyanobacteria likeS.elongatusPCC 7942 orSynechococcussp.PCC 7002 provided a unique opportunity to examine the key factors related to HL tolerance.Detailly,theS.elongatusPCC 11801 genome shares more than 80% identity with that ofS.elongatusPCC 7942.But,S.elongatusPCC 11801 shows a fast doubling time of 2.3 hours under its optimal growth conditions at 41 °C,light intensity of 1000 μmol photons·m-2·s-1and bubbling of ambient air.Similarly,the genome ofS.elongatusPCC 11802 shares 97%identity with that of its closely relatedS.elongatusPCC 11801.It has a doubling time of 2.8 h at 1% CO2and 1000 μmol photons·m-2·s-1.In addition,Synechococcussp.PCC 11901 is a robust cyanobacterial strain that can grow at 750 μmol photons·m-2·s-1,while its genome shares 96.76%identity toSynechococcussp.PCC 7002.Notably,though 99.8% genome identity is shared betweenS.elongatusUTEX 2973 andS.elongatusPCC 7942,S.elongatusUTEX 2973 could keep fast growing under HL up to 1,500 μmol photons·m-2·s-1.Recent studies have demonstrated that mutations of three genes includingatpAencoding the FoF1 ATP synthase subunit,ppnKrelated with the NAD+kinase andrpaAencoding a response regulator were mainly responsible for the HL tolerance [84,98].Excitingly,introduction of the C252Y mutation ofatpAinS.elongatusPCC 7942 dramatically increased its intracellular ATP synthase activity,photosystem II activity and tolerance to HL [85],suggesting of the universality of the HL-related elements and it might be worthy of evaluation in other cyanobacteria.In the future,as several HL-tolerant cyanobacteria have been identified,developing enough genetic tools to accelerate the gene manipulation of these species would provide new chassis for“photosynthetic cell factories”.Meanwhile,identification and introduction of HL-related genes/modules fromS.elongatusPCC 11801,S.elongatusPCC 11802 orSynechococcussp.PCC 11901 to traditional model cyanobacteria as a mean of improving HL tolerance are also worthy trying.

5.Prospective and Future Direction

Significant progresses have been achieved on deciphering the response mechanisms to HL.Based on these understanding,Qiaoet al.(2020) recently improved the production of trehalose from 4.2 g·L-1to 5.7 g·L-1when introduced the mutation C252Y of AptA inS.elongatusPCC 7942,further demonstrating the importance of the HL-tolerant chassis [100].Therefore,construction of more robust and HL-tolerant chassis would definitely contribute to the improved production of bio-chemicals in cyanobacterial cell factories.For the future studies,elucidation of new HL-tolerant mechanisms driven by new technology or from newly discovered natural HL-tolerant cyanobacteria would be important.In addition,strategies like adaptive evolution that have been demonstrated valuable and efficient in creating stress-tolerant cells in heterotrophic species such asEscherichia coli(E.coli) and yeast,should also be carried out under HL stress.Finally,with the combination of all these new findings and the development of synthetic biology tools,de novore-design of a chassis with artificial photosystem could also be a tangible focus in the future (Fig.2).

5.1.Elucidation of HL-tolerant mechanisms using new technology

Radical redesign and re-construction of the photosynthetic apparatus or its regulatory network in cyanobacteria require significant genetic engineering at genome scale.Efficient genetic manipulations tools like CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)and genome-scale modeling strategies will be important [101].Ungereret al.(2016) developed a CRISPR/Cpf1 tool to achieve marker-less knock-ins,knock-outs and point mutations inS.elongatusUTEX 2973,SynechocystisandAnabaena7120 [102].The marker-less nature of cpf1 genome editing will allow for complex genome modification that is not possible with previously existing technologies while facilitating the engineering of cyanobacteria[102].In addition,as a high-throughput genome-scale modifying platform for screening superior phenotypes,CRISPRi technology has been widely used inEscherichia coli[103–105].Recently,through a modified CRISPRi technology,Yaoet al.(2020) successfully constructed a genome-scale library ofSynechocystiswith all genes being respectively suppressed [106],providing an ideal resource to identify new genes related with HL response.In the future,libraries could be constructed for other cyanobacteria species,especially for natural HL-tolerant species.

Genome-wide photosynthetic and metabolic models are valuable in predicting photosynthetic productivity of cyanobacteria under transient conditions,providing useful directions for chassis modifications.So far,several models have been developed for model cyanobacteria likeSynechocystis[107–109],providing the mathematical metabolic model basis for other cyanobacteria,such as natural HL-tolerant species whose model yet to be established.Currently,very limited studies have utilized these models for prediction and analysis of HL stress related genes/networks.In addition,30%–60% of the total proteins were unknown or hypothetical in cyanobacteria [110],limiting the coverage and accuracy of the metabolic models.In the future,elucidating the functions of un-annotated proteins building more precise photosynthetic model and validation of the predictionsin vivowill be crucial for the construction of HL-tolerant cyanobacterial chassis.

5.2.Engineering cyanobacteria referred to natural HL-tolerant species

Fig.2.Future research directions for constructing HL-tolerant cyanobacterium.(A)The new technology-based systems for genome editing.(B)Engineering cyanobacterium refer to natural HL-tolerant species.(C) Evolutionary engineering of integrated application of ALE.ALE:adaptive laboratory evolution.(D) Systematic reconstruction of photosystem based on synthetic biology.

Several natural HL-tolerant strains have been identified.Therefore,useful information from these fast-growth and HL-tolerant strains could be obtained for directing the construction of other HL-tolerant strains from the following aspects:i)Photosystem with high throughput and high efficiency.It seems that PSII has reached the highest activity in most cyanobacterial species,whileS.elongatusUTEX 2973 exhibits more PSI,cytochrome b6f and plastocyanin content per cell which could alleviate an photosynthetic electron transport chain bottleneck[111].The increased content of electron carriers allows a higher flux of electrons through the photosynthetic electron transport chain [111].In addition,S.elongatusPCC 11802 has elevated levels of nucleotides,ATP,ADP,and AMP at high CO2,indicating better photosynthesis and ATP production[96]Therefore,a HL-tolerant strain must have a high capacity of photosystem and energy pool to bear much energy from HL.ii)High proportion of key central pathway fluxes.High carbon assimilation,more active Rubisco activity and content inS.elongatusUTEX 2973 andS.elongatusPCC 11802 than other model cyanobacterial species enable cells to fix more carbon sources at high efficiency[96,112,113].S.elongatusUTEX 2973 shows greater Calvin-Benson cycle fluxes and higher levels of key intermediate metabolites (glycolysis,photorespiration and pyruvate kinase),limited flux through the tricarboxylic acid cycle (TCA) pathway,very limited flux through the TCA and oxidative pentose phosphate pathway(OPPP)pathway which are not necessary or advantageous under all growth conditions [112,114,115].Photorespiration is necessary for growth under HL,although it is considered a carbon waste process under normal growth conditions [52,116,117].S.elongatusUTEX 2973 has a high reincorporation of the fixed carbons lost in anabolic and photorespiratory pathways in conjunction with flux rerouting through a non-decarboxylating reaction such as phosphoketolase [118].Photorespiration also fully meets the glycine and serine demand for growth [118].iii)Smart switch between energy use and storage.The synthesis of glycogen and carbon partitioning plays an important role in energy balancing during cyanobacterial growth [96].Studies showed that fast-growingS.elongatusUTEX 2973 andS.elongatusPCC 11802 can directed the high flux of carbon into biomass under suitable growth condition,allowing more flux of carbon into glycogen to store the excessive energy under suboptimal condition [96,111],although its utilization in engineering chassis yet to be made.As important primary producers and significant contributors of fixed carbon budget on earth,cyanobacteria have been present in many different environments from coast to open sea for～2.5 billion years[119],where HL is one of the key challenges for cells,it is thus fully expected that more of the natural HL-tolerant cyanobacterial species will be isolated in the future.Together with those previously identified,they constitute a valuable resource for understanding and determination of HL-related genes,modules and networks.

5.3.Laboratory-based directed adaptive evolution under HL

Although laboratory-based adaptive evolution is a timeconsuming process,it is one of the most effective strategy to obtain robust chassis with genome-wide modifications,as various studies have successfully obtained stress-tolerant strains via directed evolution [120].For example,through a continuous 94 passages for 395 days,Wanget al.(2014)achieved a 150%increase of the butanol tolerance inSynechocystiswith gradually increased butanol concentration from 0.2% to 0.5% (v/v) [121].In addition,over 802 days’ continuous passages under increasing cadmium stress,a robust strain ofSynechocystis(named ALE-9.0) tolerant to 9.0 μ mol·L-1CdSO4was successfully isolated [122].With the aid of whole genome re-sequencing,nine mutations were identified for the evolved strain compared to the WT strain,among which a large fragment deletion inslr0454encoding a cation or drug efflux system protein was found to contribute directly to the resistance to Cd2+stress [122].More importantly,overexpression of another four genes in WT significantly improved its tolerance to CdSO4[122].Moreover,the evolved ALE-9.0 strain was found to obtain cross tolerance to some other heavy metals like zinc and cobalt as well as higher resistance to high light [122].Similarly,saltand isobutanol-tolerantSynechocystischassis were respectively obtainedviaadaptive evolution [121,123],demonstrating the feasibility of this strategy in enhancing HL tolerance in cyanobacteria.Interestingly,so far,no study has focused on enhancing cyanobacterial tolerance to HLviaadaptive evolution thus it will be worthy of attempts in the future.

6.Conclusions

With the development and application of synthetic biology,dozens of bio-chemicals have been successfully synthesized using cyanobacterial chassis.More importantly,construction of HLtolerant cyanobacterial chassis will be the key for their largescale outdoor cultivation.In this review,we critically summarized the current progress on elucidating the response mechanisms of cyanobacteria towards HL and strategies to modify their tolerance.Finally,the future directions were discussed.With the development and application of synthetic biology tools,we believe more robust and HL-tolerant cyanobacterial chassis would be created in the future.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by grants from the National Key ResearchandDevelopmentProgramofChina(No.2019YFA0904600,2018YFA0903600,2020YFA0906800 and 2018YFA0903000),the National Natural Science Foundation of China (No.31770035,31972931,91751102,31770100,31901017,31901016,32070083 and 21621004),and Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(No.TSBICIP-KJGG-007).