Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. 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Various been have discussed. future been in have fuels cyanobacteria used fossil engineered be and can approaches metabolically and that by advancements limonene, functions recent production isoprene, the review, biofuel like expand this molecules and vastly In will tools biofuels. operon biology An of single synthetic generation in in the cyanobacteria approach. genes techniques for target of transformation biology cyanobacteria multiple in photosynthetic synthetic composition contain advancement engineering genetic the can and for which Simple from methods strain endeavour modification a molecules. genetic research construct biofuel tools, to increased biology of to synthetic range leads of wide development which photoau- improved a prospective manipulation of the genetic production are effortless the composition allows for genetic factories simple cell a totrophic having microorganisms prokaryotic photosynthetic Cyanobacteria, Abstract 2021 24, July 1 . Biology Indrajeet Synthetic of Tools Fuels using Sustainable Cyanobacteria of by Production Photosynthetic the in Approaches I BHU IIT 1 ki Rautela Akhil , α- ansn,suln,akns uao n at cd hc a easbttt fpetroleum of substitute a be can which acids fatty and butanol alkanes, squalene, farnesene, 1 2 n ajyKumar Sanjay and , noognccmonsuigslreeg r fgetsgicn cellular significant great of are energy solar using compounds organic into 1 1 Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. 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No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. fteedpout codn oReee l 21) h aeRScnhv nosattranslational inconstant have can RBS same plays tools the calculating (2014), RBS overexpression organisms. for same al. RBSs the et suitable in Reeve genes of different to selection or Thiel According microorganisms the manner, different in of same in product. help efficiencies native the end studies were In as These 7 the selected gene. which of efficiency. was EYFP of translation Ptrc1O the out checking by long. RBS for sequences pair 13 base RBS assessed More 22 the also coli pumps. were of (2018) efflux which strength PnrsD, metal RBS, al. PnrsB, the zinc native et check of and 20 help to cobalt evaluated nickel, the promoter (2018) by with the induced al. 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Data may be preliminary. ek stenl evsa iulrpre ee hs eut eevrfidb ie l(06 by (2016) al et Li by verified were results These gene. reporter visual al as et of serves Wendt mutants in concentration nblA (nblA) efficiency. succinate the the editing A increasing this protein as improve nonbleaching week) technique produce et 1 based (Watanabe to CRISPR mutants system site. homozygous 9 genomic 2016). elongatus producing Cyanobacteria CRISPR/Cas al., target in genome. used et the difficulty the Zerulla (2016) shows of to Its strand and nature specific the CRISPR/Cas 2015 in both is cleaves proteins. al., polyploid which which less. site associated and target (sgRNA), marker oligoploid the CRISPR RNA to is being protein repeats/ single-guide which Cas palindromic the the system, directs by short SgRNA CRISPR/Cas provided interspaced the is regularly is targetability Clustered tool biology for synthetic stands recent organism most host The the in Technique to used Based toxic widely CRISPR are is 2.4 inducer riboswitch riboswitches dependent the theophylline of only therefore, Some and intermediates, Zhang systems. 2018). metabolic cyanobacterial 2013; key al., al., et are et (Sun and Singh riboswitch, cofactor) 2008; (molybdenum Gladyshev, Moco ( SAH riboswitch (Tetrahydrofolate) riboswitch, ri- Lysine riboswitch, THF Purine element), SAMI/IV-variant riboswitch, (RFN translational and riboswitch box FMN (a-proteobacteria) SMK boswitch, SAM-II riboswitch, Glycine (SAM), (TPP)-riboswitch, S-box pyrophosphate thiamine includes cyanobacteria as in such used cobalamin synthesize which strain cosphaerawatsonii the in- in regulate work in to well cannot work used al., riboswitch also et cobalamin-dependent (Chi is riboswitches, chococcus robustness theophylline-responsive It cellular in from increases type) Apart glycogen 2010). wild of 2019). al., level of Optimised et 300% (GlgC). 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Data may be preliminary. a omdlrcoig(ol)(nlre l,21)sse o ynbcei,hwvrVsdvne al. et Vasudevan however cyanobacteria, for there system and Earlier 2014) cell, al., respectively. per al., et phase copies (Heidorn et stationary genomic gene and 4 (Engler the to exponential (MoClo) has 3 in copy cloning having copies every 7942 modular genomic PCC and and no 58 stating each drawback RBS, and (2011) was that major promoter, does 218 ensured al. the having the be et or be 6803 Griese to utilizes to cyanobacteria by PCC is gene prove epitomized it in cyanobacteria is heterologous as in This function the interest level 2011). have of case includes ploidy gene do High this strategy of Another In which gene. integration source in homologous 2017). genes strain. the of al., the the of help et sequences of the of (Lee terminator with robustness 1 place interest the NS figure of in I, affect in gene gene NS not the shown like with heterologous as cyanobacteria sites method the of neutral used genome integrating these widely the Replacing for the available in 2015). vectors is detected al., replicative recombination sites et of neutral (Ng list of the etc. number PCC shows II, are pMB1 for 3 There with Table vector it shuttle them. purposes. combining maintain research constructed and replicon stably (2018) a to consists al. selection/stress 5.2 antibiotic et PCC without are Jin plasmid used of gene 6803’s replication commonly hosts. 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Data may be preliminary. hc ucin speusr fboul Kote l,21) ilnwmn ulmlclshv been have molecules 2. 6. figure fuel Table < in many in demonstrated shown now are are Till properties which fuel yields having < 2018). molecules good al., various in of et cyanobacteria pathway intermediates biosynthetic engineered (Knoot metabolite Schematic metabolically of biofuels harvest variety a by Cyanobacteria of a be produced and 2017). precursors could efficiently sugars Woo, and as production simple and emission functions synthesize the 2009 gas and which (Melis, greenhouse for photosynthesis and energy engineering through renewable crises metabolic energy energy and solar for to sustainable candidate related of concerns alternative suitable overcome great a to been fuels potential has of cyanobacteria cyanobacteria years, recent modified In by produced molecules fuel Prominent relative close its like transformable, naturally < not are they manipulation. because genetic in plasmids process conjugal conjugation and Synechococcus tri-parental helper applied of from successfully help transmission has DNA (2015) 2015). al. non- in al., of transferred strains et conjugal be helper, (Yu rial can employs DNA which plasmids Target conjugation, replicable tri-parental strains. called other and method in well-developed Some common a by not strains 2017). is competent competency al., natural et of (Daneilla attribute This and inhibitor DNases 2007) in as al., segment EDTA et DNA using cyanobacteria. like fragment linear of strains 2011). 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Synechococcus Synechococcus ontoe xn cyanobacte- fixing nitrogen to C 92(Johnsberg 7942 PCC Synechocystis TX27 ihthe with 2973 UTEX C 92wsfirst was 7942 PCC p for sp. Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. pl el ti n ftesmls cci eqiepns aual,i ep npliain eddispersion, seed pollination, in helps it Naturally, sesquiterpenes. acyclic simplest the of one is It peel. apple α- α-Φαρνεσενε 4.3 from synthase limonene by 2017). produced was limonene strain higher cyanobacterial in transferred were P ( promoter spearmint inducible (Kiyota the pathway pyrophosphate (MEP) study from allyl phosphate dimethyl another gene 4 In limonene, erythritol of 2014). methyl precursors al., via of plant et (IPP) synthesis the pyrophosphate the isopentenyl from in and involved the gene (DMAPP) are from synthase Although that gene limonene genes enzyme. three research, synthase cloned synthase a limonene limonene In utilize by which researchers cyanobacteria. limonene DMAPP tenuifolia gene, into to and synthase transfer converted IPP limonene and MEP be diesel are plants by possess pathway for can synthesized not MEP fuel and are substitute of do isoprenoids limonene a cyanobacteria, products cyanobacteria of as In end precursors recognized The 2014). as been al., pathway. acts has et phosphate) Chucks in Limonene 4 2009; found erythritol orange. al., commonly (methyl like et is (Tracy smells Limonene fuels and plants. jet fruits in and citrus synthesized of mainly is peel molecule the isoprenoid 10-carbon a Limonene, Limonene isoprene 4.2 1.26g/l resulted study which another synthase 2016). (Meval- In isoprene MVA al, with 2014). et the combination al., (Gao with in et production combination (idi) (Bentley in isomerase enhanced, pyrophosphate was expressed tenyl yield is isoprene gene fold synthase 2.5 120 elongatus Synechococcus isoprene enzymes, system. pathway When, closed acid) a in 2012). onic production al., isoprene for et hour (Bentley 192 50 over was observed CO isoprene and of 6803 of yield addition The intermittent (2010). promoter. used isoprene PsbA2 al. produce regulated et light to Lindberg ex- the researchers by Heterologous many under reported ( IPP was of plant’s 2004). isoprene an strategy of introduced al., contain the production They et been cyanobacterial to First has (Barkley reported plant system. DMAPP been from cyanobacterial (IPP) and in have gene diphosphate IPP synthase also isoprene of isopentenyl (DXP). Cyanobacteria of inter-conversion and phosphate pression the reactions. deoxy-xylulose (DMAPP) catalyses catalysed into that enzyme diphosphate converts isomerase of di-methylallyl and series glyceraldehyde to substrate a utilises isoprenoid converted initial through MEP which further as of enzyme step pyruvate is (DXS) first and synthase Which The xylulose (G3P) 2000). 2) deoxy of phosphate (Lichtenthaler, (figure by step molecules 3 pathway catalysed final terpenoid (MEP) is of the phosphate variety pathway erythritol isoprene, a biosynthetic methyl to of with synthesis (DMAPP) synthase equipped the diphosphate isoprene are for qualifies di-methylallyl possess they However which don’t of re- Cyanobacteria composition synthesis. conversion genetic attracted isoprene the production. simple have biofuel catalyses and cyanobacteria for which rate chassis Nowadays and gene growth photosynthetic isolated fast great be isoprene. of can as of capabilities plants them higher their production the for microbial at from attention environment for gene searcher’s the cyanobac- synthase microbes and in isoprene bacteria However in emitted algae the and transferred isoprene. like synthesize eucalyptus microorganisms in not Naturally and synthesized do kudzu 2018). teria Chaves, naturally oak, 2012; like is (Melis, plants temperature which perennial higher molecule and deciduous hydrocarbon of volatile leaves a (2-methyl-1,3-butadiene), Isoprene Isoprene 4.1 ansn 37 1tiehloea13,E1-eree ly oei ln eec n a on rtin first found was and defence plant in role a plays 11-trimethyldodeca-1,3E,6E,10-tetraene) (3,7, Farnesene a nrdcdinto introduced was trc Wn ta 2016). al et (Wang C 92wsegnee o h rdcino speeb vrepesn isopen- expressing over by isoprene of production the for engineered was 7942 PCC urramontana Pueraria etaspicata Mentha yehcsi sp. Synechocystis Synechococcuselongatus 2 sn speesnhs Ip)gn engineered gene (IspS) synthase isoprene using Synechocystis6803 ne h oto fisopropyl of control the under ) etaspicata Mentha speesnhs ee(sS into (IspS) gene synthase isoprene ) C 83udrtecnrlo togpooe.Te also They promoter. strong a of control the under 6803 PCC 7 92wsgntclymdfidwt ioeesynthase limonene with modified genetically was 7942 M and oehnetelmnn rdcin Two-fold production. limonene the enhance to . irslimon Citrus spicata μ oprdto compared gg -1 β- C.Aohrrsac group research Another DCW. rgnlmnn ytaegene synthase limonene origin aatprnsd (IPTG) galactopyrenoside D μ gg Synechocystis yehcsi sp Synechocystis -1 C C il a found was yield DCW . limon Schizonepeta Lne al., et (Lin C 6803 PCC PCC . Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. qaeesnhs eewsjie oete e nyeFPo h E aha rthe or pathway MEP the of FPP mgL enzyme 11.98 key production a squalene either resulted to cyanobacterium strain Engineered joined model was phycocyanin. in gene done synthase been squalene also Inactivation has L hopene. mg production into 0.67 conversion into Squalene squalene resulted gene catalyzes 2083 which slr enzyme of (shc) that generation cyclase predicted Photosynthetic hopene group from squalene 2014). research encodes synthesized A al., is et emission. (Englund pollutant Squalene further of NADPH is CO which of from 2014). formed, molecule squalene medicine, is al., a (PSPP) of and utilizing diphosphate et condensation food presqualene step, squalene, (Englund first and cosmetics, into In molecules like petroleum converted FPP synthase. uses two squalene of by between many catalyzed instead occurs from reaction reaction two-step fuel Apart a microorganisms in as (FPP) and 2016). di-phosphate used animals al., farnesyl plants, be et by (Xu can synthesized pathways squalene MVA naturally and molecule, MEP isoprenoid via 30-carbon a is Squalene 2015). Squalene al, et 4.5 lipase (Peramuna by a DCW produced of and alkane be ADO % also FAR, 12.9 can of to Alkanes copies corresponded 2013). which seven 1200 al., overexpressed found When et was group conditions. (Wang yield research DCW alkane stress condition, of oxygenase, salt deficiency) aldehyde-deformylating 1.3% in (nitrogen and be strains reductase to cyanobacterial protein found some was carrier yield of acyl-acyl a maximum series via both al- synthesized the a expressing into are constructed over into converted alkenes coworkers by converted mainly further and pathway strains is is Wang second biosynthetic acyl-ACP aldehyde enzyme. In Fatty alkane fatty synthase (FAR). (ADO). pathway two polyketide oxigenase reductase one deformylating mainly ACP aldehyde In acyl are by now. fatty Alterna- There kanes enzyme till pollution. factories. the cyanobacteria environmental huge cell aldehydeby in and cause fatty cyanobacterial identified input which by energy been generated high produced have are a pathways be lubricants requires by-products can propane, petroleum toxic oil alkane of diesel many tively, refining gasoline, also scale include Industrial and They molecules. petroleum. manpower fuel of more constituents many major and the of one are Alkanes Alkanes 4.4 2017). al., et (Lee having gene plasmid synthase incorporating farnesene of heterologous directly production express by The to farnesene engineered was producing cyanobacteria) produce Recently started (from competent to strain gene light growth. The synthase and their dioxide farnesene for 2014). carbon source al., utilize et carbon can (Halfmann a which cyanobacteria, require to and focus farnesene. nature their in moved heterotrophic have researchers are organisms Several stated The and formed. 2017) are al., et sesquiterpenes (Tippmann produce glucose) CA, all to of Emeryville precursors as made in such which headquartered been by have pathway Biotechnologies, polymers MEP Amyris efforts solvents, have for Cyanobacteria precursor of vitamins. engineered the point company forms sucrose. cloud and jet also a products emollients, for It has renewable degC. precursor It 2017), -3 a the 2008). of al., Mcphee, forms point and it cloud et Renninger diesel’s 58) (Yoo 2016, D2 of with al., numbers compared et (cetane Yang degC -78 2010; density Keasling, energy and (K high (Peralta agent having biofuel signalling and chemical nature a in as hygroscopic acts and etc. shrci coli Escherichia nbeasp. Anabaena α- ansn rmcro ixd a on ob 4.6 be to found was dioxide carbon from Farnesene 2 sagetatraieslto fhge nutilpouto otadminimization and cost production industrial higher of solution alternative great a is 03 ggo lcrl Wn ta. 2011), al., et (Wang glycerol) of mg/g (0.38 C 19(lmnoscaoatra ile 305.4 yielded cyanobacteria) (filamentous 7129 PCC owyspruce Norway arwalipolytica Yarrowia α- -1 ansn hog eei oicto n eaoi engineering metabolic and modification genetic through farnesene qaee eet iehge hnwl tan(nln ta. 2014). al., et (Englund strain wild than higher time seventy squalene, acaoye cerevisiae Saccharomyces lnre l,20;PcosadWiae,20) en less Being 2004). Whitaker, and Pechous 2009; al., et ¨ ollner .Similarly, ). 8 Synechocystis 65m/ fguoeadfuts)(age l,2016). al., et (Yang fructose) and glucose of mg/g (6.5 yehccu elongatus Synechococcus μ nbeas.7120 sp. Anabaena gg -1 C Kgym ta. 05.Another 2015). al., et (Kageyama DCW yehccu lnau 7942 elongatus Synechococcus acaoye cerevisiae Saccharomyces C 83psessr28 eewhich gene 2083 slr possess 6803 PCC opouefreeefo sugarcane from farnesene produce to nNso punctiforme Nostoc in -1 ± yehcsi C 6803 PCC Synechocystis Co t l,2017). al., et. (Choi . gLi days. 7 in mg/L 0.4 μ / ansn n1 days 15 in farnesene g/L a rw nsl stress salt in grown was C 92(naturally 7942 PCC 05 ggof mg/g (0.57 β- C 73102 PCC uui of subunit nwhich in mutant Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eotdt rdc 59 C nrclua ii n .%etaellrfe at cd Enrsmeet (Eungrasamee acids (CBB) fatty basham free Benson- extracellular gene 9.6% calvin- encoding and of lipid synthetase genes intracellular 2020). in glpD protein DCW al., engineered and 35.9% carrier were produce LXS acyl-acyl to genes rbc with reported study, encoding corresponding engineered synthetase another w/DCW acyl-ACP In was 34.5% and 2019). which to al., up strain observed et the was (Eungrasamee production in lipid acetyl-coA mg/l/d Maximum encoding synthetase protein acids). genes 41.4 carrier fatty targeted acyl-acyl They free and strategy. hydrolysis) of novel (phospholipid (recycling free A a lipase of using engineered synthesis), synthesis group secretion acids research acid (fatty for A carboxylase fatty 2012). expressed enhanced Jones, over for and was 197 (Ruffing 6803 yield gene PCC to low very encoding up resulted thioesterase which increased acids and fatty and was gene Liu encoding yield synthetase secretion synthesis. acyl-ACP acid lipid enhanced fatty elongatus the The Synechococcos for gene. modified genetically thioesterase engineered be also genetically in 0.13% can co-workers lipid of cyanobacterium lowest on the a In effect from condition, a its ranged in In which observe 1990). 7.24% lipids to researchers. accumulate of al., limited to many et ability were by their (Wada nutrients) reported in (important conditions been phosphorus cyanobacteria. environmental selected has and in normal conditions productivity nitrogen in stress of nutrient lipids supply in study, neutral production accumulate petroleum lipid not great cyanobacteria their do a to CO due they chains, environmental acids fatty fatty fix but prominent cyanobacterial alkyl to and A of converted capture transesterification long reaction. are the to transesterification is of (TAGs) capacity by (FAEEs) production Triacylglycerides consisting esters biodiesel ethyl for 2019). molecules approach acid biological fatty al., fuel and (FAMEs) et esters prominent (Pandey methyl acid the requirements energy of for one substitute are acids Fatty acids Fatty 4.7 pathway. 2018). biosynthesis al., isobutanol et with (Miao strain engineered producing metabolically (3M1B) been 1-butanol from has gene 2018) (Kivd) decarboxylase cyanobacterium al., study et pathway. a pathway. Miao acid In this keto 2017, by 2- al., mixotrophic by synthesized done under et are 2009) be produced can acids was al., the isobutanol study, isobutanol which amino from of et pathway, of synthesis another chain separated mg/L Ehrlich biological In branched was 298 The the Isobutanol Mainly solvent. 2013). from a al., 2011). genes et conditions. as al., (Varman heterologous alcohol mixotrophic condition et oleyl two and by of (Lan poly autotrophic medium expression of produced in production the presence was isobutanol the for synthesize butanol in engineered can 1- enhanced was mg/L was 6803 activity 13.6 PCC Trans-enoyl-coA butyryl-coA. (drop tag. A to substitute 2012). histidine as coA al., used crotonyl- et be reduces (Peralta into can pathway engine and production It of butanol 2012). modification 1- any dependent al., A) without elongatus et gasoline (Co-enzyme hydrocarbons (Lu as CoA value petroleum introduced importance of group energy variety great research higher a has its for molecules, to fuel) due carbon in purpose four fuel of for consisting additive alcohol branched-chain a Isobutanol, Isobutanol 4.6 C 92 nti aha,teoeaetcl-cArdcae(e)wrsa rtndonor proton as works (ter) reductase coA treponemadenticola- pathway, this In 7942. PCC irclu sp Microcoleus Saccharomycescerevisiae C 92wsegnee o h rdcino reftyaisb ncigout knocking by acids fatty free of production the for engineered was 7942 PCC Synechocystis Kmre l,21) pr rmntrllpdsnhssadapyn stress applying and synthesis lipid natural from Apart 2017). al., et (Kumar . atccu lactis Lactococcus yehcsi PCC6803 synechocystis Ya ta. 07,adcaoatra(tuie l,20,Miao 2009, al., et (Atsumi cyanobacteria and 2017), al., et (Yuan siltras. nbeas. irclu p,adNso sp. Nostoc and sp., Microcoleus sp., Anabaena sp., Oscillatoria C 83wseeooosyepesdwt an with expressed washeterologously 6803 PCC 2 lhuhcaoatrapseslpdboytei pathway, biosynthesis lipid possess cyanobacteria Although . ( .lactis L. 9 ihcdnotmzday-clcrirprotein carrier acyl-acyl optimized codon with hc eutdi niouao n 3-methyl- and isobutanol an in resulted which ) Synechocystis shrci coli Escherichia C 83 oie tanwas strain Modified 6803. PCC nbeasp Anabaena ± 4mgL 14 -1 ( Lue l,2011). al., et (Liu otemaximum the to . .coli E. α- ketoisovalerate Synechococcus Synechocystis Synechocystis (Atsumi ) varied Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. tui . u .Y,Ek,E . akn,S . ule,T,&La,J .(00.niern the (2010).Engineering C. J. Liao, & T., de- Buelter, reductase/alcohol to aldehyde D., dioxide three S. of carbon Hawkins, comparison of by M., recycling coli E. genes. Escherichia photosynthetic hydrogenase Eckl, in Direct pathway Y., biosynthetic T. isobutanol (2009). Wu, C. S., J. Atsumi, Liao, cyanobacte- & in W., rhythms isobutyraldehyde. circadian Higashide, of transfor- S. study DNase genetic Atsumi, the a Improved to with bioluminescence (2017). association of F. Application in ria. L. fragments (2000). R. &Marins, DNA C. C., linear Andersson, F. using C. 7942 Lanes, PCC B., elongatus inhibitor. B. Synechococcus S. of Heterologous Martens, of mation Control V., the D. for 6803. Promoters Almeida, PCC Light-Induced sp. Evaluating Synechocystis (2017). in A. in efficiency C. Expression photochemical Peebles, Gene Increased & (2016). C., C. S. D. Albers, sink. Ducat, sucrose & engineered M., D. an Kramer, via B., cyanobacteria Kachel, that W., B. relationships Abramson, personal paper. or this interests in financial reported work competing References the known influence no to have appeared they have could that declare authors The interests competing any in of interest. or Declaration of English conflict in no form, declare same authors The the holder. copyright in the elsewhere of published consent its work statement be written the elsewhere, Ethical not the where publication authorities will without for responsible electronically it the consideration including accepted, by language, under if explicitly other not or and tacitly is out, and carried and authors was published all by been approved research not is and publication has Varanasi work submitted IIT(BHU) for This the India (MHRD), by Development provided and project Resource Declaration grant Human seed of the Ministry acknowledge the facility. SK to grateful aid. are financial AR and Indrajeet efficient developing and bioreactors photo Acknowledgements cyanobacterial utilizing scalable of interest research designing of processes. the of downstream increasing butanol metabolite scope of economic of and is chances and pathway are there alkanes there biosynthetic Currently squalene, years production. the forthcoming limonene, biofuel engineering In isoprene, biology. by synthetic like yield of interest. molecules tools good of fuel and gene in sites, of produced cyanobacteria fuels binding expression have by heterologous ribosome neutral researchers the efficient promoters, carbon the now like attracted for of Till toolboxes tools has variety into CRISPR/Cas and it insights wide phototrophic terminators provides manipulation, a riboswitches, requirements, biology genetic of Synthetic nutritional cyanobacteria. in production CO limited of simplicity photosynthetic from their chemicals the to value-added of emerged for Due other have because researchers composition. Cyanobacteria years of sources. genetic recent attention energy simple in renewable accomplishments and noteworthy and factories nature’s nature sustainable cell the creating of suitable for one is as utilized photosynthesis be of can process which the by energy solar Harvesting Perspectives Future and Remarks Concluding ehd Enzymol. Methods itcnlg eerhadInnovation and Research Biotechnology auebiotechnology Nature ple irbooyadbiotechnology and microbiology Applied , 305 527-542. , 2 s fsnhtcbooytoshssmlfidmtblcengineering metabolic simplified has tools biology synthetic of Use . , 27 (12)1177. ln n elPhysiology Cell and Plant , itcnlg progress Biotechnology 1 10 1,123-128. (1), , 85 3,651-657. (3), , , 57 33 1) 2451-2460 (12), 1,45-53. (1), Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. io . og . hn,L,Ci . hn . hn,W 22) alrn ynbcei sanew a as cyanobacteria Tailoring (2020). W. Zhang, & L., Chen, astaxanthin. J., of Cui, synthesis efficient L., highly Savage, Zhang, for & X., platform phyla. . Song, . prokaryotic . J., throughout M., found L. Diao, Oltrogge, pumps G., carbon T. inorganic Laughlin, J., are E. DABs , Dugan, C., (2019). Dynamic Blikstad, F. (2014). I., S. D. A. S. Flamholz, Golden, J., & J. J., proteins. Desmarais, Pogliano, aviation clock J., Hasty, A-1 circadian G., cyanobacterial Jet Dong, the with J., of Selimkhanov, fuels L., localization bioderived M. Erb, potential E., of S. compatibility Cohen, The (2014). J. Donnelly, kerosene. CRISPR/Cas12a & Transcription-Coupled Cell-Free J., and (2021). C. W. Chuck, engineering J. Improvement Lee, Promoters. metabolic & (2017). Cyanobacterial M., M. by Prototyping J. H. Park, for Woo, 7942 R., & Assay Y. PCC Shin, . N., elongatus . . Y. Choi, Y., Synechococcus Kim, photobioreactor. in Y., a Um, CO2 in M., S. for production from Lee, scalable interference S., production H. CRISPR squalene Kwak, Y., dCas12a-mediated of J. Wang, A cyanobacteria. Y., in S. CRISPRi-dCas12a: Choi, engineering theophylline-responsive metabolic (2020). a and M. Adopting genes H. 2351-2361. multiple elongatus (2019). Woo, of Synechococcus X. & repression in Lu, Y., metabolism & S. glycogen G., Choi, of Luan, Self- understanding C., (2016). and Qiao, W. regulation H., J. PCC7942. flexible cyanobacterium Golden, Sun, for unicellular & S., the S., riboswitch Zhang, of S. X., plasmid Golden, endogenous M., Chi, (2017). small L. L. a Pieper, 7942. Alfonta, pANS, PCC E., & elongatus on R. Synechococcus based London, . vectors M., . . shuttle Go, R., replicating A., Schwarz, Taton, E., organism. Y., photoautotrophic Nagar, Chen, a A., of Bakhrat, code M., genetic the Heltberg, Expanding M., Friedman, Y., cyanobacteria. Chemla, in synthesis isoprene Engineering process (2018). improve A. 2059-2069. to &Melis, strategies E., production: J. and Biohydrogen Chaves, promoters (2015). W. LacI-repressed routes. D. of microbial Lee, through & analysis J., efficiency and Y. Design Lee, K., Chandrasekhar, (2014). promoter. P. tac cyanobacterium. the Lindblad, a in & in regions T., -35 DNA-looping and Heidorn, -10 D., the Camsund, of Spacing (1984). pathway J. Chemistry. acid Storella Biological mevalonic and of the M., of Erfle isoprene. expression J., to Heterologous Brosius partitioning carbon (2014). endogenous A. emis- enhances &Melis, cyanobacteria isoprene A., in and Zurbriggen, uptake K., dioxide F. Bentley, carbon microorganisms. for dose–response photosynthetic process Comparative by Diffusion-based photobioreactors bioengineering two-phase (2020). (2012). gaseous/aqueous from M. A. in I. isomerase sion &Melis, &Axmann, diphosphate K., D., isopentenyl F. Dienst, II Bentley, Cyanobacteria. 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(1), , 592 , Journal 9 (12), (9), Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. io . s,A,Fky,Y,Eia . iaoi .(07.Apiaino RSRitreec for interference CRISPR of Application (2017). T. Hisabori, 7120. & PCC sp. S., Anabaena cyanobacterium Ehira, multicellular heterocyst-forming Y., the Fukaya, of engineering A., metabolic Isu, metabolism. cellular A., sense Higo, to RNA using RNAs: Riboswitch 22 Kr (2008). S., M. B12. T. Sasso, vitamin Henkin, J., of variants U. chemical Kudahl, different A., use Holzer, algae , eukaryotic D., and A. Cyanobacteria Lawrence, (2016). E., In Stensj K. functions. P., Helliwell, novel Press. 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Fleming, free S. and &Jantaro, of P., biosynthesis levels Lindblad, acid significant 6803. A., fatty the 6803 Incharoensakdi, enhances via PCC R., cycling duction Miao, sp. acid K., fatty Synechocystis acids. (2014). Eungrasamee, free fatty P. (2020). and free Lindberg, secreted S. cycle and & &Jantaro, CBB lipids P., J., in intracellular involved Lindblad, Bergquist, genes A., K., overexpressing Incharoensakdi, Stensjo, 6803. K., K., PCC Eungrasamee, J. sp. for Synechocystis S. sites in Ubhayasekera, binding squalene of 6803. B., ribosome Production PCC Pattanaik, and promoters E., sp. of Englund, Synechocystis Evaluation cyanobacterium (2016). unicellular P. the reports Lindberg, in & applications F., biotechnological Liang, oxide. diterpenoidmanoyl E., plant Englund, the engineering of Metabolic production (2015). P. for (2014). Lindberg, 6803 S. & , PCC &Marillonnet, B., sp. Hamberger, . Synechocystis R., . a . Miao, of D., J., of J. Andersen-Ranberg, plants. 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(1), cetfi reports Scientific isnhtcptwyo ieetfe oeue ncaoatra ehleyhio 4-phosphate erythritol Methyl cyanobacteria; in molecules fuel different of pathway Biosynthetic : elcmn fnurlst ihgn fitrs GI:Tre eegt netdi h place the in inserted gets gene Target (GOI): interest of gene with site neutral of Replacement : C ytei biology synthetic ACS Environments urn pno nbiotechnology in opinion Current itcnlg journal Biotechnology , 379 , 5 4,881-899. (4), 8132. , , 7 2,13. (2), , , htsnhssrsac protocols research Photosynthesis 32 5 3,207-212. (3), 1) 195 (12), , 14 irsuc technology Bioresource ple irbooyadbiotechnology and microbiology Applied 6,1800496. (6), , 45 18 1-7. , , 216 p.2323.Hmn rs,Totowa, Press, Humana 273-293). (pp. 1040-1048. , irsucsadBioprocessing and Bioresources α- ansn nmetabolically in farnesene , 101 Microbiology cetfi reports Scientific 1,465-474. (1), ol ora of Journal World , 162 Journal (5), , 4 Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. al :Pouto ffe oeue ygntclyegnee cyanobacteria engineered genetically by molecules cyanobacteria fuel of of strains Production different 6: for Table followed strategies transfer DNA 5: Table commercially available plasmids Integrative plasmid 4: replicative Table available cyanobacteria commercially in of used List promoters 3: foreign Table and native approach of biology List synthetic 2: for Table used organisms host Cyanobacterial 1: Table Geranyl tables dehydrogenase. GGPP- of Pyruvate diphosphate; List PDH- Farnesyl diphosphate; FPP- Presqualene bisphosphate; PSPP- Geranyl diphos- diphosphate; Isopentenyl GPP- IPP- geranyl synthase; isomerase; methyl diphosphate; IPP Isoprene Ipi- ME-cPP- Hydroxymethylbutenyl ISPS- diphosphate; phate; allyl HMBPP- synthase; Dimethyl DMAPP- synthase; kinase; reductase; ME-cPP HMBPP HMBPP CDP-ME IspH- IspF- IspG- IspE- cyclodiphosphate; phosphate; methylerythritol; phos- erythritol-2,4- diphosphocytidylyl methylerythritol erythritol CDP-ME- Methyl diphosphocytidylyl MEP- synthase; CDP-MEP- reductoisomerase; CDP-ME DXP IspD- DXR- 5-phosphate; phate; Xylulose Deoxy DXP- synthase; 19 Posted on Authorea 24 Jul 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162714404.47090375/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. synthetic-biology in-the-photosynthetic-production-of-sustainable-fuels-by-cyanobacteria-using-tools-of- updated.docx Table file Hosted vial at available https://authorea.com/users/427348/articles/531525-approaches- 20