Cross-Feeding between Cyanobacterium Synechococcus and Escherichia Coli in Articial Autotrophic-Heterotrophic Co-Culture System Revealed by Integrated Omics Analysis

Background: The light-driven consortia consisted of sucrose-secreting cyanobacteria and heterotrophic species capable of producing valuable chemicals have recently attracted signicant attention, and are considered as a promising strategy for green biomanufacturing. In a previous study (Zhang et al, 2020, Biotechnol Biofuel, 13:82), we achieved a one-step conversion of CO 2 through sucrose derived from cyanobacteria to ne chemicals by constructing an articial co-culture system consisting of sucrose-secreting Synechococcus elongateus cscB + and 3-hydroxypropionic acid (3-HP) producing Escherichia coli ABKm. Analysis of the co-culture system showed that cyanobacterial cells were growing better than its corresponding axenic culture. To explore the underlaid mechanism and to identify the metabolic modules to further improve the co-culture system, an integrated metabolomics, transcriptomic and proteomic analysis was conducted. Results: We rst explored the effect of reactive oxygen species (ROS) on cyanobacterial cell growth under co-culture system by supplementing additional ascorbic acid to scavenge ROS in CoBG-11 medium. The result showed cyanobacterial growth was obviously improved with additional 1 mM ascorbic acid under pure culture; however, cyanobacterial growth was still slower than that in the co-culture with E. coli, suggesting that the better growth of Synechococcus cscB + might be caused by other factors more than just ROS quenching. We then investigated the intracellular metabolite levels in cyanobacteria using LC-MS based metabolomics analysis. The results showed that metabolites involved in central carbon metabolism were increased, suggesting more carbon sources were utilized by cyanobacteria in the co-culture system, which illuminating that enhanced photosynthesis attributes to the higher CO 2 availability produced from co-cultivated heterotrophic partner. To further explore the interaction based on cross-feeding and metabolite exchange, quantitative transcriptomics and proteomics were applied to Synechococcus cscB + . Analysis of differentially regulated genes/proteins showed that the higher availability of carbon, nitrogen, phosphate,


Introduction
Cyanobacteria with the capability of producing organic matter from CO 2 by using solar energy, have attracted increased attention as environmentally friendly and sustainable "microbial cell factories" for the production of carbohydrate feedstocks to support traditional fermentation processes 12 . Take the sucrose, an easily fermentable feedstock, as example, several cyanobacterial species are capable of synthesizing and secreting sucrose as an osmolyte under appropriate environmental stimuli, such as osmotic pressure 3 , and this process can be sustained over a long period of time and at higher levels than that from plant-feedstock such as sugarcane and beet 4,5 . However, puri cation of sucrose from cyanobacterial cultivation supernatant is costly and the system is easily contaminated, which creates barriers to any scale-up cultivation 6 . In addition, any application of photosynthetic cell factories in scaleup facilities is always restricted by challenges from harsh environments, suggesting that the adaptability and compatibility of cyanobacterial cell factories should be further improved to facilitate the industrialscale biomanufacturing 7 . In recent years, increasing evidences suggested that the exchange of essential metabolites between microorganisms could be a crucial process that can signi cantly affect growth, composition and the structure stability of microbial communities in nature 8, 9 . In aquatic environments, the ecological interaction between photo-autotropic and heterotrophic species is based on cross-feeding and metabolite exchange 10 . In this case, the photo-autotrophs excreted material ranging from targeted photosynthetic intermediates such as glycolate, osmolytes and fatty acids, and extracellular polymeric substance, to the products of cell lysis that can include sugars, proteins, lipids and nucleic acids 11,12 . In exchange, heterotrophic species are thought to provide essential micronutrients, such as vitamins, amino acids and bioavailable trace metals, necessary to maintain high photosynthetic productivity 9 . In addition, the positive effects on the autotrophs were also observed, which might be attributed to the decrease of oxidative stress by heterotrophs through reactive oxygen species (ROS) scavenging 13,14 . Inspired by the symbiotic system commonly found in nature, increasing efforts have been made in recent years to design arti cial routes of metabolite interchange in order to construct new symbiotic systems with high e ciency and stability 15,16 .
The light-driven arti cial consortia consisted of sucrose-secreting cyanobacterium and heterotrophic species have recently attracted signi cant attention as the alternatives for the utilization of sucrose from cyanobacteria 17 . For example, Ducat et al. constructed a co-culture system consisting of the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter as Synechococcus 7942) and the heterotrophic bacterium Halomonas boliviensis, in which the growth of H. boliviensis was supported by sucrose produced by S. elongatus 7942 18 . In another study, Li et al. designed a co-culture system with the sucrose-secreting S. elongatus 7942 and three different yeasts to mimic lichen and research the interaction between the autotrophic and heterotrophic strains 13 . More recently, Liu et al. constructed a coculture system composed of S. elongatus 7942 and E. coli to produce isoprene and extended the fermentation time of co-cultivation was extended from 100 h to 400 h by adjusting the inoculation ratio between S. elongatus 7942 and E. coli, in which the production of isoprene was increased sevenfold to 0.4 g/L compared to the axenic culture of E. coli 19 . In addition, cyanobacteria other than model species were also utilized, for example, Zhang et al. constructed a microbial consortium consisting of the fastgrowing cyanobacterium Synechococcus elongatus UTEX 2973 recently identi ed (hereafter as Synechococcus 2973) which are capable of growing under high light and temperature 20 , as well as E. coli to sequentially produce sucrose and then the platform chemical 3-hydroxypropionic acid (3-HP) from CO 2 21 . All these studies enlightened us that light-driven co-culture system could be a promising strategy for future CO 2 based biomanufacturing.
To construct light-driven co-culture systems with high e ciency, it is necessary to fully understand the metabolic mechanism underlaid the interaction between autotrophs and heterotrophs. Although several previously studies have showed that the cyanobacterial cell growth could be improved in co-culture system 13 , the mechanism is yet to be determined. Moreover, while it is fully expected that the mechanism involves more than just single gene, or even single metabolic module, so far only a few studies utilized global-based omics techniques to explore the interaction mechanism at multi-level of RNA, protein and metabolite [22][23][24] . Due to the complexity of co-culture structure, the challenge to study on the interaction mechanism is also increased, integrated omics analysis could be a good approach to obtain a "panorama" of cells in the co-culture systems and reveal novel insights into the biological mechanism 25 .
For example, Amin et al. analyzed the signaling and interaction between diatom and associated bacteria through integrated metabolite and transcriptomic analysis, in which the tryptophan and indole-3-acetic acid were determined as the key signaling molecules involving in the complex exchange of nutrients 26 , demonstrating that the approach of integrated transcriptome, proteome and metabolome should be adopted to explore microbial interactions in the co-culture systems.
In our previous study, Zhang et al. 21 constructed an arti cial co-culture system composed of the sucrosesecreting strain Synechococcus elongatuscscB + and the sucrose-utilizing and 3-hydroxypropionic acid (3-HP) -producing strain E. coli ABKm. The system was able to produce ~68.29 mg/L 3-HP in 7 days. In spite of the one-step sucrose utilization co-culture system was successfully constructed, productivity and stability of the co-culture systems remain challenging. In this study, an integrated proteomics and transcriptomics approach was employed to analyze the metabolic responses of cyanobacteria to the heterotrophic partner in the arti cial co-culture system, which will be valuable to identify the metabolic modules involved in e ciency and stability of the co-culture system and apply them as potential engineering targets to further optimize the system, as well as for guiding cultivation optimization.

Strains and culture conditions
The sucrose-secreting strain SynechococcuscscB + (derived from Synechococcus elongatus UTEX 2973) and the sucrose-utilizing and 3-HP-producing E. coli ABKm strain reported in our previous study were used to construct co-culture system 21 . SynechococcuscscB + was cultivated under 100 μmol photons m -2 s -1 in an illuminating shaking incubator (HNYC-202T, Honour, Tianjin, China) at 130 rpm and 37 °C or on BG-11 agar plates in an incubator (SPX-250B-G, Boxun, Shanghai, China) 27 . E. coli ABKm strain were grown on LB medium or agar plates with appropriate antibiotics added to maintain plasmids at 37 °C in a shaking incubator (HNY-100B, Honour, Tianjin, China) at 200 rpm or in an incubator, respectively. Co-culture medium (hereafter as CoBG-11) was used to construct co-culture system according to the previous study 21 , in which 150 mM NaCl, 4 mM NH 4 Cl and 3 g/L 2-[[1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl] amino] ethanesulfonic acid (TES) were supplemented into the BG-11 medium. The pH value was adjusted with NaOH to 8.3.
For construction of co-culture system, the exponential phase SynechococcuscscB + (OD 750 ≈1.0) was collected and inoculated into 25 mL of CoBG-11 and grown at 30 °C for 48 h to an OD 750 of 0.5. E. coli was cultivated in CoBG-11 with 1 g/L sucrose for 48 h, and then the cells were collected and resuspended in deionized water and inoculated into the 25 mL SynechococcuscscB + culture grown on CoBG-11. To separate two species in the co-culture system, the dialysis bag (diameter is 36 mm, molecular weight cutoff is 14 kDa, respectively, biosharp, Hefei, China) was used. The E. coli ABKm was incubated in the dialysis bag, while the SynechococcuscscB + was incubated outside in the ask. The pre-treatment of dialysis bags was according to a previous study with some modi cations 28 . Brie y, the dialysis bag was cut into small pieces of appropriate length (approximately 10 cm), which were boiled for 10 minutes with a large volume of 1 mmol/L EDTA (pH 8.0). And then the dialysis bags were boiled with distilled water for 10 minutes for twice. The prepared dialysis bag was autoclave sterilized before using.
The cell density was measured at OD 750 using a UV-1750 spectrophotometer (Shimadzu, Kyoto, Japan).
The co-cultivated SynechococcuscscB + was counted by a hemocytometer under a microscope (BX43, Olympus, Shinjuku, Tokyo, Japan) after series dilution.

LC-MS based metabolomics analysis
Liquid chromatography-mass spectrometry (LC-MS) based targeted metabolomics was performed according to the protocol described previously 29 . Cells (5 OD 730 unit) were harvested at 48 h via centrifugation at 7380 rpm for 5 min at 4 °C (Eppendorf 5430R), quenched, and extracted rapidly with 900 μL of 80:20 methanol/water (v/v; -80 °C pretreated) and then frozen in liquid nitrogen. Intracellular metabolites were extracted via the freeze/thaw cycle for three times. The aforementioned extraction process was repeated with another 500 μL 80:20 methanol/water (v/ v). The supernatant was combined and ltered through a 0.22 μm syringe lter. The solvents were removed using a vacuum concentrator system (ZLS-1, Hunan, China), and 100 μL of ddH2O was added and mixed well. LC-MS analysis was conducted using an Agilent 1260 series binary HPLC system equipped with a Synergi Hydro-RP (C18) 150 mm × 2.0 mm ID, 4 μm 80 Å particle column (Phenomenex, Torrance, CA, U.S.A.), and an Agilent 6410 triple quadrupole mass analyzer equipped with an electrospray ionization (ESI) source. Data were acquired using the Agilent Mass Hunter work-station LC/QQQ acquisition software (version B.04.01), and chromatographic peaks were subsequently integrated via the Agilent Qualitative Analysis software (version B.04.00). All data of metabolomic pro ling was rst normalized by the internal control and the cell numbers of the samples. Each condition analysis consisted of four biological replicates and three technical replicates.

Transcriptomic analysis of cyanobacterial responses to E. coli in co-culture system
Considering the characteristics of transcriptomics technology and the accuracy of transcriptomics data, dialysis bags were used to separate cyanobacteria and E. coli to construct the co-culture system. For transcriptomic analysis, 5 OD 750 of co-cultured and ascorbic acid treated axenic SynechococcuscscB + were collected respectively for extracting RNA samples; meanwhile, the same amount of SynechococcuscscB + cultivated under axenic was used as control. The transcriptomics analysis was conducted by GENEWIZ (Suzhou, China). There three biological replicates for each sample, and two statistic parameters which are fold change>1.5 and Q-value (fdr or padj)≤0.05 were used to determine differentially regulated genes.

Quantitative proteomics analysis of cyanobacterial responses to E. coli in co-culture system
The same weight of four-day co-cultured strains were sampled for proteome analysis. The samples were enzymatically digested by trypsin, following marked by isobaric tags for relative and absolute quanti cation (iTRAQ) technique, the samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The axenic Synechococcus 2973 cells with same incubation time were used as control. The technical service and data of quantitative proteomics were provided by BGI (Shenzhen, China). Three biological replicates for each sample were used. In the case of unmatched biological replicates, two statistic parameters, fold change>1.2 (the average ratio of the nine comparison groups) and P-value<0.05 (t-test of nine comparison groups) were used to screen differentially regulated proteins.

Quantitative real-time PCR analysis
For RNA extraction, the 2 OD 750 cells of Synechococcus cscB + under axenic culture and co-culture were collected and centrifuged at 7,830 rpm and 4 ℃ for 5 mins. The total RNA samples were extracted using Direct-zol™ RNA Miniprep kit (ZYMO RESEARCH, CA, USA) according to the instruction, and then reverse transcribed as cDNA template using HiScript ® II Q RT SuperMix for qPCR (+gDNA wiper) reagent (Vazyme, China). The quantitative real-time PCR (qPCR) reactions were performed according to the methods described previously 30 . Brie y, the 10 L reaction system was composed of 5 L of 2  34,35 , the analysis showed that SynechococcuscscB + grew better with additional 1 mM ascorbic acid in CoBG-11 compared with 0.1 mM ascorbic acid supplementation, while cell growth was inhibited after three days when supplemented 2 mM ascorbic acid (Fig. 1A). Consistently, the analysis showed that H 2 O 2 content was signi cantly decreased with ascorbic acid added (Fig. 1B); however, it is unclear why the cells growth was arrested when 2 mM supplementary ascorbic acid led to almost no detectable H 2 O 2 at 4 days. More importantly, although cyanobacterial growth was obviously improved with additional 1 mM ascorbic acid under pure culture, cyanobacterial growth was still slower than that in co-culture with E. coli, suggesting that the better growth of SynechococcuscscB + might be caused by other factors more than just ROS quenching.
3.2 Analysis of key metabolites in S. elongateus cscB + during co-cultivation by target LC-MS metabolomics As the stability and productivity in the co-culture system was dependent on the cyanobacterial sucrose production, intracellular levels of key metabolites within Synechococcus cscB + cells were investigated. LC-MS based metabolomics approach, which has been employed previously to comparatively analyze cellular metabolism in the engineered cyanobacterial strains 29,36 , was applied to compare co-cultivated and pure cultural Synechococcus cscB + . As shown in Fig. 2, twenty-one metabolites of cyanobacterial metabolism involve in glycolysis, amino acid, and the citric acid (TCA) cycle were chemically classi ed. Comparative analysis showed that the intracellular contents of FBP, F6P, E4P, R5P and acetyl-CoA were increased, suggesting more carbon sources were utilized in co-cultivated Synechococcus cscB + . Five amino acids, lysine (Lys), serine (Ser), valine (Val), alanine (Ala) and phenylalanine (Phe) were found with signi cant up-regulation during co-cultivation condition. Three metabolites involved in TCA cycle including citric, malate and succinate, were also showed signi cant increases in co-cultivation condition.
One possible explanation for the increased metabolites in glycolysis, amino acid, and TCA cycle is that increasingly available CO 2 , possibly from the respiration of heterotrophic cells, contributes to cyanobacterial better cell growth during co-cultivation, consistent with a previous nding that enhanced CO 2 xation and oil production in co-culturing green algal Chlorella and yeast Saccharomyces cerevisiae system 37 . The results were also consistent with a more recent study which found salt stress redirect the xed CO 2 toward sucrose production rather than biomass and glycogen accumulation in engineered Synechococcus 2973 38 . In addition, the results of LC-MS also implicated that the enhanced CO 2 xation could be used as an engineering target for further improving sucrose production in the co-cultivated cyanobacteria by modulating sucrose production pathway.

Analysis of cyanobacterial metabolic responses to E. coliin co-cultivated S. elongateusby transcriptomics
For the interaction based on cross-feeding and metabolite exchange, early studies have shown that heterotrophic bacteria could also be involved in providing essential micronutrients, such vitamins, amino acids and bioavailable trace metals, necessary to maintain high photosynthetic productivity 39 . To explore the mechanism underlaid the increased cell growth of co-cultivated cyanobacteria, and to determine the factors necessary for the stability and fermentation performance, transcriptomics between pure cultural cyanobacteria (C) and co-cultural cyanobacteria with E. coli (D) was applied to analyze the interaction mechanism of cyanobacteria responses to the heterotrophic partner in co-culture system. With a cutoff of 1.5-fold change and a p value of statistical signi cance less than 0.05, we found 120 genes up-regulated and 104 genes down-regulated as a result of co-cultivation, respectively. The reliability and accuracy of the transcriptomics data was independently veri ed by real-time quantitative PCR (qRT-PCR) (Suppl Table S1), the correlation coe cient R 2 was 0.9086 (Fig. S1), indicating the transcriptomics data collected in this study is of very high accuracy. The analysis showed that a large fraction of up-regulated transcripts was a liated with photosynthesis and oxidative phosphorylation (25%), signal transduction and membrane transport (13%), translation (10%), genetic information processing (6%), metabolism of cofactors and vitamins (3%) role categories (Fig. 3A, Suppl (M744_12800) were down-regulated in our study, which likely due to the reduced ROS content in coculture system 13 . The down-regulated transcripts in co-cultivated Synechococcus cscB + compared with under axenic condition were also demonstrated (Suppl Table S3). our nding that the down-regulation of these four genes that the better growth of Synechococcus cscB + in co-culture system was partially attributed to the quenching of ROS by heterotrophic partner E. coli ABKm. In a previous study, Vance et al. found that the phospholipid/cholesterol/gamma-HCH transport system permease protein (MlaE) was down-regulated after exposure to a high bisphenol A concentration, which might inhibit phospholipid transport, and subsequently altered the spontaneous diffusion of the membrane to eventually caused membrane damage 47 . Interestingly, the relative expression of M744_01095 (mlaE) was also increased in co-cultivated Synechococcus cscB + , which also suggested that ROS induced membrane damage was relieved by the presence of the heterotrophic partner.
In cyanobacteria, the secretory (Sec) pathway is critical for proteins transportation across the plasma membrane and thylakoid membrane 48 . The core of translocase in Sec pathway is a protein channel assembled by heterotrimeric membrane protein complex SecYEG and ATPase SecA oligomers, SecA is used as a molecular motor 49 . It was estimated that 82% of translocated proteins in Synechocystis 6803 contain a Sec signal peptide 50 . The expression of M744_13645 (secE) was up-regulated in co-cultivated Synechococcus cscB + . In addition, the relative expression of M744_09155 (yidC) was increased in cocultivated Synechococcus cscB + as YidC protein mediates integration of membrane integral proteins in bacteria and thylakoid membrane 51 . The increased expression levels of secE and yidC were consisted with the phenotype of better cyanobacterial growth as translocase is responsible for the insertion of the photosystem integral membrane proteins into the thylakoid membrane in cyanobacteria 52 .

Analysis of cyanobacterial metabolic responses to E. coliin co-cultivated S. elongateusby quantitative proteomics
The low correlation between mRNA and protein expression has been found and well discussed in previous studies, which might be caused by the widespread post-transcriptional regulation mechanism 53,54 . For example, Nie et al. found that correlation of mRNA expression and protein abundance was affected at a fairly signi cant level by multiple factors related to translational e ciency 55 . In order to fully identify the interaction mechanism in the co-culture system, the quantitative iTRAQ proteomics was used to analyze cell responses of Synechococcus cscB + adapt to E. coli in co-culture system. Three Synechococcus cscB + samples from the co-culture (E1, E2, E3) and three from the axenic culture (C1, C2, C3) were collected after cultivation of 96 h, respectively, and the differential pro les of proteins in  (Fig. 4A). Most of the identi ed proteins were with good peptide coverage, ~89% of the proteins were with more than 10% of the sequence coverage and ~87% were with more than 20% of the sequence coverage (Fig. 4B). Among the functional categories, the "general function prediction only" was the top detected functional category, representing 13.43% of all the identi ed protein (Fig. 4C). This result is consisted with the previous nding that approximately 45% of proteins in the cyanobacterial genome are hypothetical proteins 56 . Other frequently detected functional categories included "translation, ribosome structure and biogenesis" (9.42%), "amino acid transport and metabolism" (8.67%), "posttranslational modi cation, protein turnover, chaperones" (8.51%), "signal transduction mechanism" (7.17%), "carbohydrate transport and metabolism" (6.51%).
3.5 Cyanobacteria responses to co-culture systemdeciphered by proteomics As demonstrated in the transcriptomics analysis that the large fraction (25%) of transcripts involved in photosynthesis and oxidative phosphorylation were signi cantly increased during co-cultivation, the differentially expressed proteins involved in the energy metabolism pathway was also identi ed (Suppl Table S4). In co-cultivated Synechococcus cscB + , the increase of protein abundances for energy metabolism enzymes, such as ferredoxin (PetF, M744_01325), phycobiliproteins terminal rod linker (CpcD, M744_11425), photosystem II reaction center H (PsbH, M744_01910), photosystem II D1 protein (PsbA, M744_00850), NAD(P)H-quinone oxidoreductase subunit 4 (NadhD, M744_05920), and NAD(P)H-quinone oxidoreductase subunit 5 (NadhF, M744_01470) were observed, suggesting that more NADPH and ATP generated from photosynthesis 57 , which is likely due to the elevated C and/or N availability compared with the axenic control, as discussed above. Meanwhile, increased protein abundance of the lightindependent prochlorophyll reductase subunit B (ChlB) (M744_07280), which catalyzes the conversion of prochlorophyll to chlorophyll a 58 , was also found, suggesting that cyanobacterial photosynthesis might be improved during the co-cultivation. These results are well consistent with our ndings based on the transcriptomics analysis.
Nitrogen metabolism, either from nitrate or ammonium, governs the turnovers of the macromolecules that regulate metabolic pathways, eventually affecting energy production and carbon skeleton 59 . Through the quantitative proteomics analysis, three nitrate/nitrite transport system ATP-binding proteins of M744_10450 (NrtB), M744_10455 (NrtC), and M744_10460 (NrtD) and two ferredoxin-nitrite reductases (M744_10440 and M744_07195) were found up-regulated in the co-cultivated Synechococcus cscB + , suggesting that the nitrite uptake in co-cultivated Synechococcus cscB + was enhanced. The ammonium is incorporated into carbon skeletons through glutamine synthetase (M744_02210), which was also found up-regulated in co-cultivated Synechococcus cscB + . Signi cant up-regulation in the nitrogen uptake and assimilation were evident with higher photosynthesis and better cyanobacterial growth during the cocultivation condition.
Phosphorus is a vital nutrient for cyanobacterial growth, which impacts the synthesis of cyanobacterial extracellular polymeric substances and also appears to induce signi cant changes in the synthesis of polysaccharides, as well as membrane lipids 60 . In the co-cultivated Synechococcus cscB + , the proteins involved in phosphate transport system, including M744_04030 (PstA), M744_04035 (PstB), M744_04025 (PstC), M744_04020 (PstS) and M744_04015 (SphX), were found up-regulated by 2.08-, 2.12-, 3.80-, 2.67-and 4.75-fold, respectively. The up-regulation of all four phosphate transporters in the co-cultivated Synechococcus cscB + might be due to the increased consumption of Pi in the form of NAPDH or ATP, which contributes to further cell growth. Consistently, the increased transcripts level of M744_04015 (sphX) and M744_04030 (pstA), were also found at transcription level. Aside from dissolved inorganic phosphorus, dissolved organic phosphorus is used by cyanobacteria via alkaline phosphatase 60 . Two alkaline phosphatases (M744_09635 and M744_11635) in the co-cultivated Synechococcus cscB + were found up-regulated by 2.89-and 1.40-fold, respectively, suggesting that the cyanobacteria were able to acquire more phosphorus for cell growth during the co-cultivation condition 40 .
Two up-regulated proteins M744_05990 and M744_04340 annotated respectively as xylose-5phosphate/fructose 6-phosphate phosphotransketolase (Xfp) and pyruvate-ferredoxin/ avodoxin oxidoreductase (Por), were identi ed in the co-cultivated Synechococcus cscB + . Xfp plays a key role in glycolysis, catalyzing the conversion of X5P or F6P to acetyl phosphate 61 , while Por is responsible for the oxidation process of pyruvate to generate acetyl-CoA 62 . The up-regulation of Xfp and Por indicated CO 2 xation might be enhanced in the co-cultivated Synechococcus cscB + , well-consistent with the increased acetyl-CoA content in the metabolomic analysis discussed above. In addition, three bicarbonate transporters, including M744_08440 (CmpB), M744_08445 (CmpC), and M744_08450 (CmpD) were also found down-regulated in the quantitative proteomics data, also consistent with the transcriptomic analysis. The cmp operon (cmpA, cmpB, cmpC, cmpD) in Synechococcus 7942 has been con rmed to encode a component of the ABC-type HCO 3transporter BCT1, and its transcription was activated at low CO 2 concentrations 63, 64 . The down-regulation of these three bicarbonate transporters indicated that the concentration of CO 2 in the co-culture system might be higher than that under pure culture conditions, due to the fact that E. coli ABKm might secret CO 2 to the system, which was also found in transcriptomics.
The exchange of essential micronutrients, such as vitamins, amino acids and bioavailable trace metal, from heterotrophic bacteria to cyanobacteria during co-cultivation was observed above at the transcript level. Pathway enrichment analysis of the proteomic data indicated that Synechococcus cscB + had maximized the uptake and utilization of Fe 3+ and thiamine to improve cell growth during the co- Moreover, the down-regulation of ve proteins related to oxidative stress, including Hli protein (M744_01810 and M744_11065), fur family transcription regulator (Fur) (M744_05500 and M744_12665), and monothiol glutaredoxin (M744_10930) were found in the co-cultivated Synechococcus cscB + by our quantitative proteomics analysis. The two Hli proteins were also identi ed in transcriptomic analysis. The transcription regulator Fur, as an iron uptake regulator, is responsible for controlling the gene expression of siderophore biosynthesis and iron transport 70 . In previous studies, monothiol glutaredoxin was proved to protect against oxidative stress by regulating iron homeostasis 71 .
The crosstalk between controlling iron homeostasis and defending against ROS was previously con rmed in E. coli, demonstrating that the lack of iron regulation may lead to oxidative stress 72 . Thus, the down-regulation of these ve proteins indicated the positive effect on cell growth during the cocultivation was attributed to the decrease of oxidative stress in Synechococcus cscB + by a heterotrophic cells capable of ROS scavenging, which was also consistent with the our previous nding that the gene expression of three types of catalases, including hydroperoxidase I (katG), hydroperoxidase II (katF), and hydroperoxidase III (katE) were signi cant induced in E.coli during the co-cultivation condition 21 .

Conclusion
In our previous study, we constructed a one-step conversion system from CO 2 , and sucrose synthesized and secreted by cyanobacteria to a ne chemical 3-HP by including a sucrose-secreting cyanobacterial Synechococcus cscB + and a 3-HP producing E. coli ABKm in a co-culture system maintaining for one week, and achieved a 3-HP production of 68.29 mg/L 21 . However, the stability and 3-HP productivity in the co-culture system are still low, probably due to the low cyanobacterial biomass and sucrose productivity in the co-culture system, and even the low metabolite exchange between the two partner cells in the system 73 . To further improve the functional performance of the co-culture system, an integrated omics analysis was conducted in this study to determine the interaction mechanism between cyanobacterium Synechococcus and E. coli.
The stability in the autotrophy-heterotrophy co-culture system dependents on the cyanobacterial growth and sucrose production. In this study, the decreased level of several oxidative stress related proteins was found in transcripts level (M744_11065, M744_01810, M744_03995 and M744_07160) and proteomics level (M744_01810, M744_11065, M744_05500, M744_12665 and M744_10930), suggesting the possible reduced oxidative stress in co-cultivated Synechococcus cscB + . In addition, the increased expression of transcript related to phospholipid/cholesterol/gamma-HCH transport system (M744_01095), and decreased expression of many transcripts involved in ribosomal proteins (M744_13675, M744_13670, M744_05205, M744_00735, M744_05210, M744_12320, M744_05195, M744_05180, M744_05185), tRNA synthetases (M744_03935, M744_5340) and RNA binding protein (M744_12800) were found in cocultivated Synechococcus cscB + , also suggesting that ROS induced membrane damage was relieved by the presence of the heterotrophic partner. All these nding illuminated us that the antioxidative system of E. coli could be further enhanced through overexpressing the major ROS-scavenging enzymes, for example superoxide dismutase, catalase, glutathione peroxidases and thioredoxin 74 , to improve cyanobacterial cell growth and productivity during co-cultivation. Previous studies have shown that heterotrophic species could provide essential micronutrients, such as vitamins, amino acids and bioavailable trace metals, necessary to maintain high photosynthetic productivity in various co-culture systems 9 . In this study, the higher availability of carbon, nitrogen, phosphate, calcium, Cu 2+ , Fe 3+ and cofactors in co-cultivated Synechococcus cscB + during co-cultivation were identi ed by the integrated metabolomics, transcriptomics and proteomics analysis, which therefore with great promise as the potential targets to improve the fermentation performance of the co-culture system consisted with photoautotrophic and heterotrophic species.
Light condition is critical for the cell growth of cyanobacteria through photosynthesis 75 , especially under co-cultivation, as the cell concentration increases, light-shading caused by the heterotrophic species might reduce cyanobacterial exposure to light and thus the photosynthetic activity 76 . Meanwhile, the enhanced photosynthesis and oxidative phosphorylation identi ed by the integrated omics also illuminated light condition might be further optimized to improve the stability and e ciency of arti cial co-culture system. More importantly, cyanobacteria are often inhibited by ROS produced from the imbalance between light absorption and light utilization during the process of photosynthesis 77 .
Although in co-culture system, the inhibition of oxidative pressure on cyanobacteria can be reduced by the heterotrophic species 13,21 , the growth of heterotrophic species can be inhibited by ROS when exposed to high densities of cyanobacteria in the light 17,78 . Considering the light availability may in uence the cross-feeding metabolites between phototrophic and heterotrophic species including oxygen and carbon dioxide through photosynthesis and respiration, and also affect the oxidative pressure on the E. coli strains due to the photosynthesis 25 , the optimal light conditions could also be critical to the high cell growth in the co-culture system.
In conclusion, the results showed that many metabolic changes, including enhanced photosynthesis, oxidative phosphorylation, and essential micronutrients, were discovered at multiple levels, not only the ROS scavenging, might be responsible for the better cell growth of Synechococcus cscB + during cocultivation (Fig. 5). We thus proposed that the metabolic modules related to the ROS quenching, carbon metabolism, nitrogen metabolism, Pi transport, metal transport and co-factors biosynthesis could be potential engineering targets to further improve stability and fermentation performance in this co-culture system.
Declarations Figure 1 Analysis of the effect of quenching ROS on cultivated cyanobacterial cell growth by adding ascorbic acid.
Cell growth curves of Synechococcus cscB+ (A) and H2O2 content (B) in co-culture system and axenic culture with additional ascorbic acid. compared with axenic culture condition; (B) Heatmaps of metabolomics pro les in Synechococcus cscB+ under co-culture and axenic culture condition. Each color on the heatmap corresponds to a concentration value. The higher the concentration, the darker the color (red represents the increase, and green represents the decrease).