| Cyanobacteria are a class of photoautotrophic prokaryotes that can use sunlight as energy to convert carbon dioxide(CO2)into organic matters.Compared with higher plants,cyanobacteria have the advantages of higher photosynthetic efficiency,faster growth rate,no requirement for arable land,and easier genetic accessibility.With the emergence of fast-growing cyanobacteria which grow as rapid as that of the heterotrophic microorganism Saccharomyces cerevisiae,cyanobacteria have received extensive attention in the research and application of photosynthetic carbon fixation.Synechocystis sp.PCC 6803(designated as Synechocystis 6803)is the first model cyanobacterium that has been completely genome-sequenced,and is also commonly used as a photosynthetic chassis cell for chemicals production from CO2.Benefited from the development of cyanobacterial metabolic engineering and synthetic biology,engineered Synechocystis 6803 strains were reported to convert CO2 into more than a dozen of chemicals including ethanol,isopropanol,and isoprene.However,strain Synechocystis 6803 is only able to grow under low light intensity,and the optimum light intensity is between 50 to 100 μmol m-2 s-1.Consequently,the strain grows slowly,with a doubling time up to 8 h,and the yields of the target chemicals are low.Therefore,it is of great significance to screen Synechocystis 6803 mutant that grows rapidly under high light intensity,with high biomass and strong stress tolerance,and study the underlined molecular mechanism.This will promote the development of cyanobacterial photosynthetic CO2 fixation.In this dissertation,the photosynthetic physiology of a mutant of Synechocystis 6803(designated as HL6803),which was obtained in our laboratory’s previous experiments and could grow normally under moderate light intensity of 250 μmol m-2 s-1,was analyzed and characterized.The tolerance of HL6803 to environmental factors such as high light intensity,high temperature,high salinity and high alkalinity were also assessed.Through comparative genomic and transcriptomic analysis with Synechocystis 6803 wild type(designated as WT6803),the mechanism of rapid growth of HL6803 under high light intensity was proposed,and its capacity of using CO2 for production of target chemicals was tested.The main findings are as follows:1.Growth experiment showed that HL6803 was able to grow normally under the high light intensity of 900 μmol m-2 s-1,and accumulated more than twice the biomass of WT6803.Determination of the content of major photosynthetic pigments showed that the content of chlorophyll a in HL6803 was 40%higher than that of WT6803.In addition,the photosynthetic oxygen evolution rate of HL6803,measured by a photosynthetic oxygen evolution instrument,was nearly 5 times higher than that of WT6803.The chlorophyll fluorescence kinetics of photosystem Ⅱ(PSⅡ)and photosystem Ⅰ(PSI)were measured by chlorophyll fluorometer.Compared with WT6803,the light saturation point of HL6803 increased by 56%.the ability of selfphotoprotection through non-photochemical quenching improved by 2.65 times,and the photosynthetic efficiency of PSⅡ and PSⅠ increased by 94%and 90%,respectively.2.Comparative genomics analysis revealed that HL6803 had five mutated genes that have not been reported in other fast-growing cyanobacteria.These included sll1533 encoding a type Ⅳ fimbrial protein PilT2,slr0947 encoding a response regulator RpaB,sll0020 encoding an ATP-dependent Clp protease regulatory subunit ClpC.sll1434 encoding a penicillin-binding protein 1B,and slr0914 encoding a hypothetical protein with a transmembrane helix structure,respectively.These mutated genes are likely involved in the rapid growth of HL6803 under high light intensity.Efforts for introducing the mutations identified in these five genes into WT6803 were made by using CRISPR/Cas9 method,but no correct mutant was obtained to date.3.High temperature,high salinity and high alkalinity stress experiments showed that HL6803 exhibited a stronger tolerance to high temperature and high salinity than that of WT6803.Comparative transcriptomic analyses of HL6803 and WT6803 were conducted under high light intensity,high light intensity and high temperature,high light intensity and high salinity stress conditions.A total of 77 genes in HL6803 were differentially expressed under high light intensity conditions(36 up-regulated,41 down-regulated),a total of 9 genes in HL6803 were differentially expressed under high light intensity and high temperature conditions(2 up-regulated,7 down-regulated),whereas a total of 78 genes in HL6803 were differentially expressed under high light intensity and high salinity conditions(34 up-regulated,44 down-regulated).These differentially expressed genes in HL6803 were associated with different metabolic pathways.The up-regulated genes were mainly involved in light energy capture,electron transport chain,high light stress tolerance,organic matter synthesis and intracellular energy consumption,while the down-regulated genes were mainly involved in decomposition of carbohydrates,amino acids and other organic materials.This suggests that HL6803 regulates the balance of intracellular energy and material metabolism to improve its stress tolerance.4.To evaluate the capacity of HL6803 for producing organic materials,the biosynthetic pathway from CO2 to β-farnesene was constructed based on its endogenous mevalonate(MEP)pathway.Introduction of a farnesene synthase AaFS derived from Artemisia annua into HL6803 led to production of β-farnesene.The MEP pathway was optimized by introducing an isopentyl diphosphate isomerase AaIDI.As a consequence,the production of β-farnesene reached 6.6±0.1 mg/L in 7 days,and the highest productivity reached 2.0±0.4 mg L-1 day-1,the highest among the cyanobacterial βfarnesene production studies reported to date.This indicates that HL6803 has the potential to be developed into a light-driven efficient carbon-fixing cell factory.5.To test whether the electrons in HL6803 can be exported extracellularly,an exogenous conductive protein OmcS from Geobacter sulfurreducens PCA was introduced into HL6803.Unexpectedly,the expression of OmcS in HL6803 did not improve cell growth nor its photochemical activity.On the contrary,expression of OmcS in Synechococcus elongatus UTEX 2973(designated as Syn2973),a cyanobacterium capable of rapidly growing under high light intensity,led to extracellular export of electrons.The photochemical activities of PSⅡ and PSⅠ were greatly improved,and the intracellular content of ATP and NADH were also increased by about 30%and 60%,respectively.Comparative transcriptomic analysis showed that the up-regulated genes in Syn2973 with OmcS were mainly involved in light energy absorption and photosynthetic carbon fixation.The dysfunction of OmcS in HL6803 suggests that HL6803 and Syn2973 have great differences in energy metabolism and material metabolism.In conclusion,this study found that HL6803 has higher photosynthetic physiological activity,stronger energy metabolism regulation capability,and better environmental adaptability.HL6803 can be used as an alternative chassis cell for the development of light-driven carbon-fixing cell factories.Comparative genomic and transcriptomic analyses revealed specific mutations and possible intrinsic mechanisms for regulation of intracellular energy and material metabolism balance of HL6803.This provides targets and references for further studying the mechanism of HL6803 for tolerating high light intensity,and may facilitate the improvement of light utilization efficiency of photosynthetic organisms. |
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