Enhancing Bioethanol Production by Deleting Phosphoenolpyruvate Synthase and ADP-Glucose Pyrophosphorylase, and Shunting Tricarboxylic Acid Cycle In Synechocystis Sp. PCC 6803

Enhancing Bioethanol Production by Deleting Phosphoenolpyruvate Synthase and ADP-Glucose Pyrophosphorylase, and Shunting Tricarboxylic Acid Cycle In Synechocystis Sp. 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PCC 6803 E-Bin Gao, Penglin Ye, Haiyan Qiu, Junhua Wu, Huayou Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-193538/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The outstanding ability of directly assimilating carbon dioxide and sunlight to produce biofuels and chemicals impels photosynthetic cyanobacteria to become attractive organisms for the solution to the global warming crises and the world energy growth. The cyanobacteria-based method for ethanol production has been increasingly regarded as alternatives to food biomass-based fermentation and traditional petroleum-based production. Therefore, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 to synthesize ethanol and optimized the biosynthetic pathways for improving ethanol production under photoautotrophic conditions. Results: In this study, we successfully achieved the photosynthetic production of ethanol from atmospheric carbon dioxide by an engineered mutant Synechocystis sp. PCC 6803 with over-expressing the heterologous genes encoding Zymomonas mobilis pyruvate decarboxylase (PDC) and Escherichia coli NADPH-dependent alcohol dehydrogenase (YqhD). The engineered strain was further optimized by an alternative engineering approach to improve cell growth, and increase the intracellular supply of the precursor pyruvate for ethanol production under photoautotrophic conditions. This approach includes blocking phosphoenolpyruvate synthetic pathway from pyruvate, removing glycogen storage, and shunting carbon metabolic flux of tricarboxylic acid cycle. Through redirecting and optimizing the metabolic carbon flux of Synechocystis , a high ethanol-producing efficiency was achieved (248 mg L -1 day -1 ) under photoautotrophic conditions with atmospheric CO 2 as the sole carbon source. Conclusions: The engineered strain SYN009 ( ∆slr0301/pdc-yqhD , ∆slr1176/maeB ) would become a valuable biosystem for photosynthetic production of ethanol and for expanding our knowledge of exploiting cyanobacteria to produce value chemicals directly from atmospheric CO 2 . Biotechnology and Bioengineering Cyanobacteria Pyruvate Phosphoenolpyruvate Ethanol production Glycogen synthesis Tricarboxylic acid cycle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Due to the high ability of directly integrating atmospheric CO 2 with sunlight into biomass, cyanobacteria have been increasingly proposed as one of the most promising biosystems for the solution to the global warming crises and the world energy growth [1]. Cyanobacteria are autotrophic prokaryotes that perform similar photosynthesis as higher plants [2]. However, compared to the traditional plants, cyanobacteria possess several advantages, including the simple inorganic nutrient requirement, the tolerant growth on non-arable land, and powerful genetic manipulation [3], which render photosynthetic cyanobacteria as attractive organisms for the direct production of important chemicals from atmospheric CO 2 through metabolic engineering methods [4]. Over the years, an enormous amount of the engineered cyanobacteria has been successfully developed to produce various industrial relevant chemicals, such as ethylene [5], isoprene [6], ethanol [7], isobutanol [8], acetone [9], and p -coumaric acid [10]. Ethanol is one of the major renewable biofuels that are a worldwide focus of main concern. Commercially, ethanol production is mostly based on the fermentation of starch or agricultural crops as feedstock [7]. The over-exploitation of this food-based raw material presents a significant bottleneck for expanding ethanol production. To avoid competition with the world food supply or agricultural land, the cyanobacteria-based method for ethanol production has been increasingly regarded as alternatives to biomass-based fermentation. In recent years, a lot of efforts have been made to realize ethanol production by engineered cyanobacteria. The first production of ethanol was successfully achieved in Synechococcus elongatus PCC 7942 by expressing pyruvate decarboxylase (PDC) and alcohol dehydrogenase II (ADH II) from Z. mobilis [11]. Soon afterward, Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) was genetically engineered by expressing these two enzymes to produce ethanol, and obtained double the ethanol yield compared to S. elongatus PCC7942 strain [12]. To further improve ethanol production, many desirable strategies were adopted to optimize abiotic and biotic factors that have effects on cyanobacterial cell growth and metabolisms. For example, overexpressing the ethanol-producing steps and blocking the production of storage polymers (glycogen and PHB) were performed in Synechocystis 6803 to increase ethanol production [13]. Recently, another example has been significantly shown to improve the production of ethanol by engineered cyanobacteria with enhanced cell growth through overexpressing the Calvin-Benson-Bassham (CBB) cycle enzymes [14]. However, genetically engineered cyanobacteria-based biosystems still faced many challenges for ethanol production applications, such as redirecting carbon flux to the desired product instead of cellular biomass, promoting cell growth, and balancing the co-factor levels due to metabolic consumption of an introduced biosynthetic pathway. To enhance the ethanol production by cyanobacteria, more efficient and rational biosynthetic pathways need to be established through metabolic engineering. In this study, considerable attention is paid to achieve the ethanol production in the model cyanobacterium Synechocystis 6803 by genetically integrating the genes encoding Z. mobilis PDC and E. coli YqhD into the Synechocystis 6803 chromosome. To investigate the potential of optimizing metabolic pathways for improving ethanol productivity, the engineered cyanobacterial strains were genetically modified in a stepwise approach via inhibiting phosphoenolpyruvate pathway from pyruvate, removing glycogen storage, and shunting carbon metabolic flux of the tricarboxylic acid cycle. These approaches led to the high-efficient ethanol production directly from solar energy and atmospheric CO 2 under photoautotrophic conditions, and significantly contribute to enhancing the biological synthesis of the desired carbon-based biofuels. Results Determining the metabolic pathways for the ethanol production To achieve photosynthetic production of ethanol in Synechocystis 6803, it is essential to rationally construct and optimize an exogenous biosynthetic pathway for redirecting carbon fluxes towards ethanol. Thus, the precursor intracellular level and the catalytic pathway efficiency are generally considered as two regulating factors for high-efficient production of ethanol. In cyanobacteria, the pyruvate and acetyl-CoA are two important intermediate metabolites, which are naturally accumulated in cells, and successfully exploited by several studies as the starting precursors to be converted into ethanol and other products. To evaluate which intermediate precursor is more efficient for ethanol production, we measured the intracellular content of the primary metabolites in the wild-type Synechocystis 6803 strain. As shown in Fig. S1, the intracellular concentration of pyruvate reached up to an average of 1.05 µmol g -1 (dry cell weight), which was approximately 3 times that of acetyl-CoA. This result indicated that Synechocystis 6803 possesses a high potential ability to produce ethanol by using pyruvate as the starting precursor rather than acetyl-CoA. The major pathway of ethanol production was constructed by expressing two enzymes, Z. mobilis PDC and E. coli YqhD . As shown in Fig. 1, the pyruvate was directly converted to acetaldehyde and CO 2 by PDC, and subsequently, acetaldehyde was reduced by YqhD to generate ethanol and NADP + . The Cu 2+ inducible promoter PpetE was initially selected to drive the expression of the exogenous genes. The promoter PpetE and the codon-optimized genes were integrated into the neutral site ( slr0168 ) of Synechocystis 6803 genome to generate the initial strain SYN001 ( ∆slr0168/pdc-yqhD, PpetE ) (Fig. 2A, Fig.2B). The resulting transformants were maintained in liquid BG-11 medium with the addition of 50mg/L spectinomycin. HPLC analysis showed that ethanol accumulated up to a yield of 230 mg L -1 (OD 730 ≈0.64) in the strain SYN001 after 7 days of photoautotrophic growth (Fig. 3A, Fig. 3B). According to the published studies [15, 16], the strength of the promoter PpetE was categorized as “medium”, which led to relatively low expression levels of the targeted genes. This enabled us to hypothesize that the mRNA expression levels of the pdc and yqhD gene were potentially correlated with ethanol production yield. To overcome the potential effects of possible low expression of the ethanol biosynthesis genes, we substituted the ‘medium’ promoter PpetE with the light-sensitive and strong promoter PpsbA2s to construct the second producer SYN002 ( ∆slr0168/pdc-yqhD , PpsbA2s ) (Fig. 2A, Fig.2B). After 7 days of cultivation, HPLC analysis demonstrated that 474 mg L -1 (OD 730 ≈0.64) of ethanol was accumulated in the culture medium, which was 2-fold higher than that of strain SYN001. These results demonstrated that enhancing the gene expression was required in Synechocystis for the high-efficient production of ethanol. This is also consistent with previous studies where the relatively high expression levels of biosynthetic genes were required to improve the production of the desired chemicals. Eliminating phosphoenolpyruvate pathway from pyruvate Due to continuous production and consumption by several metabolic pathways, the intermediate pyruvate undergoes dynamic fluctuation at the intracellular level. This rendered us attempt to lessen the competitive consumption of the precursor pyruvate by inhibiting the catalytic activity of phosphoenolpyruvate synthase encoded by the slr0301 gene of Synechocystis 6803 genome. Theoretically, disruption of the slr0301 gene would metabolically block the catalytic conversion of the pyruvate to phosphoenolpyruvate, and then could increase the endogenous carbon flux from pyruvate to ethanol. To test if the catalytic inhibition of phosphoenolpyruvate (PEP) synthase activity could contribute to ethanol formation, the engineered strain SYN003 ( ∆slr0301/pdc-yqhD ) was constructed by transforming the plasmid pBE03 inculcating the slr0301 knockout cassette into the wide-type Synechocystis strain. The ethanol-producing assay was performed to compare the yield of the newly engineered strain SYN003 with the strain SYN001 and SYN002. The ethanol yield was improved to 600 mg L -1 (OD 730 ≈0.64) after 7 days of photoautotrophic growth. The yield of the strain SYN003 was approximately 2.6-fold and 1.3-fold higher than that of SYN001 and SYN002, respectively. This result gave an implication that catalytic inactivation of phosphoenolpyruvate synthase led to the level increase of the precursor pyruvate, and then boosted the ethanol formation. To determine whether disruption of the slr0301 gene has negative effects on the photoautotrophic growth of the engineered Synechocystis strain SYN003, the time-courses of OD 730 were performed to compare the growth pattern of the strain SYN003 and SYN004 ( ∆slr0301 ) with the wild-type strains. Obviously, no differences were observed between the engineered strains and the wild-type strain (Fig. 4), indicating that the deletion of the slr0301 gene has no significant effects on the physiological activities of Synechocystis 6803. Based on these results, rational optimization of the pyruvate consumption pathway might provide a useful strategy that can contribute to effectively increase the ethanol biosynthesis in the engineered strain of Synechocystis 6803. Blocking glycogen synthesis pathway To enhance photosynthetic carbon flux towards chemical production by cyanobacteria, removing the glycogen biosynthetic pathway would become a helpful strategy for the generation of biofuels and chemicals. In this study, to test if inactivation of glycogen synthesis contributes to the ethanol production in the phosphoenolpyruvate synthase-deficient strain ( ∆slr0301/pdc-yqhD ), we also performed complete inhibition of the AGPase activity by disrupting the slr1176 gene of Synechocystis 6803 genome. The engineered strain SYN007 ( ∆slr0301/pdc-yqhD , ∆slr1176 ) was constructed by transforming the plasmid pMD-slr1176-Ω inculcating the slr1176 gene knockout cassette into the strain SYN003. The ethanol yield in this double-knockout strain reached up to 689 mg L -1 (OD 730 ≈0.57) under the photoautotrophic conditions. The strain SYN007 showed about 1.5-fold ethanol yield compared with the starting strain SYN002. Similar to the strain SYN003 and SYN004, no remarkable differences were shown between the engineered SYN007 and the wild-type strain (Fig. 4). These data confirmed that complete inactivation of glycogen synthesis can be beneficial for the direct conversion of the fixed carbon in photosynthesis to the desired ethanol. This is also consistent with the previous studies that blocking the glycogen synthesis pathway can shift excess carbon from the glycolytic pathway and pentose phosphate pathway to a favorable biosynthetic pathway. Metabolic interventions of the TCA cycle It has been reported that the major portion of the fixed carbon in Synechocystis 6803 was metabolically consumed by the aerobic TCA cycle, which is one of the central carbon metabolisms in cyanobacteria [17]. Those carbon reserve metabolites produced from the TCA cycle are of great interest because they are efficient substrates for the production of ethanol or other biofuels. Thus, in the next optimization step, we attempted to improve ethanol production by shunting the metabolic route of Synechocystis TCA cycle metabolism. The metabolic intervention of the TCA cycle was conducted by over-expressing NADP-dependent malic enzyme ( mae B) from E. coli . The maeB gene was introduced into the slr1176 site of the strain SYN003, establishing SYN009 ( ∆slr0301/pdc-yqhD , ∆slr1176/maeB ). After 7 days of photoautotrophic growth, the strain SYN009 was able to generate ethanol, which the production yield reached up to 1.09 g L -1 (OD 730 ≈0.7) (Fig. 3B), and showed more than 1.6-fold improved ethanol yield compared with the strain SYN007. To our surprise, under photoautotrophic conditions, the strain SYN009 displayed normal growth and propagated steadily over culture time, and showed 30% higher cell density than the wide-type strains (Fig. 5A). To indicate the hypothesis that the engineered strains produce higher yield of ethanol over long-term growth, the strain SYN009 was cultivated for up to 14 days. As expected, the ethanol accumulation was increased by 30% and improved to 1.3 g L -1 (OD 730 ≈1.37) at 14 days of photoautotrophic culture. Interestingly, the ethanol yield in strain SYN009 was increased dramatically during the growth stage of days 0-4. During each of these days, the ethanol productivity was greater than 200 mg L -1 day -1 (Supplementary materials Fig. S2). The highest ethanol productivity reached 248 mg L -1 day -1 (OD 730 ≈0.70) at the 4th day. After day 4, the ethanol yield showed a slight increase, but the ethanol productivity significantly decreased to less than 100 mg/L/day. This may be that the insufficient supply of CO 2 and light limits ethanol production of the strain SYN009 under photoautotrophic conditions. Taken together, these data indicated that a basic photo-biosystem for ethanol production was developed in our experiment through metabolic engineering optimization, which contributes to redirecting the metabolic carbon flux towards ethanol production. Discussion It has been reported that the metabolic imbalance of endogenous metabolism and the biosynthetic pathway often limits the desired chemical productivity and yield by microbial systems [18]. To enhance the carbon flux towards ethanol production, it is necessary to counterbalance the contradictory relationship between endogenous metabolism and the biosynthetic pathways. Therefore, we focused on the genetically engineering of a cyanobacterial strain for enhancing the capacity of metabolic flux toward the pyruvate, an important intermediate metabolite for ethanol production. For this purpose, we developed several modified metabolic bypasses to boost the intracellular supply of pyruvate in the engineered Synechocysits harboring the ethanol-producing pathway. Our integrative approach led to successful improvement of the ethanol yield and productivity via stepwise metabolic engineering, e.g., disruption of consuming pyruvate pathways and reorientation of the carbon flux of the TCA cycle towards pyruvate. In the glycolytic pathway, phosphoenolpyruvate (PEP) is catalyzed by pyruvate kinase into pyruvate, and reversibly, the pyruvate is converted into PEP through the activity of PEP synthase (PpsA). This means that PpsA could be an attractive target for pyruvate forming metabolic bypass from PEP. In Synechocystis 6803, PpsA is encoded by the slr0301 gene. To the best of our knowledge, no experimental evidence has to date been reported on the potential effects of the disruption of Synechocystis PpsA on the ethanol production. To test if this could be the case in Synechocystis , we constructed the strain SYN003 by deleting the PEP synthase coding locus: slr0301 . As expected, with the inhibition of PEP synthase activity, the ethanol production yield in the strain SYN003 showed significant increase when compared to the strain SYN001 and SYN002. Moreover, the cell growth of the PEP synthase-deficient Synechocystis is normal and similar to that of the wide-type strains. These results suggested that the optimized metabolic bypass can regulate the strain SYN003 intracellular metabolism to improve the pyruvate supply for ethanol production. It can be suggested that deletion of the PEP synthase contributed to the yield of ethanol production by repression of PEP synthesis. To further improve the ethanol yield in our study, an alternative strategy is to perform the complete inhibition of glycogen synthesis pathway. Glycogen is generated from CBB cycle, and considered as one of major storage components for carbon resources in cyanobacteria. Significantly, if Synechocystis 6803 cells lack the ability of glycogen synthesis, they will show an overflow of carbon metabolism leading to the excretion of pyruvate [19]. This effect may be hijacked for product formation by introducing a pyruvate-utilizing reaction such as ethanol production [20]. Several studies have been conducted to increase the ethanol production in cyanobacteria by deleting the glycogen synthesis pathway [13, 21]. To check if this could also be the case for Synechocystis , we optimized the strain SYN003 by knocking out the glgC : slr1176 gene encoding AGPase to obtain the glycogen-deficient strain. As expected, the strain SYN007 produced more ethanol than the strain SYN003. The marked increase of ethanol yield in the strain SYN007 strongly confirmed that complete inhibition of glycogen synthesis could contribute to the ethanol production. Also, this is the first investigation of the effect of combinatorial inhibition of PEP synthase and glycogen synthesis on ethanol production. It can be hypothesized that the production of the increased level of ethanol in the stain SYN007 could be caused by the repression of the cell growth and glycogen storage. Although the enhancement of ethanol production was observed in the strain SYN007, it would be necessary to preform metabolomics and proteomics analysis for further understanding the regulating mechanisms or metabolic network. In this study, considerable research attention has been made to remove the potential bottlenecks in ethanol biosynthetic pathway. As the intercellular concentration of pyruvate, the major intermediate precursor, is metabolically controlled by several endogenous pathways. Traditionally, it holds the view that the pyruvate was mainly generated from the PEP through pyruvate kinase [7]. However, recent studies have shown that a carbon flux is significantly channeled via the TCA cycle through the malic enzyme to pyruvate, rather than generating pyruvate directly from the ATP-generating reaction catalyzed by pyruvate kinase [22, 23]. Thus, the metabolic interference of the TCA cycle is suggested as an alternative way to boost ethanol production [24]. For this purpose, a previous study has been performed in an attempt to investigate the effects of an endogenous gene slr0721 encoding malic enzyme ( me ) in Synechocysitis 6803 on the ethanol production [25], suggesting that the ethanol production was associated with the optimal level of malic enzyme activity in Synechocysitis 6803. To test whether the potential effects of exogenous malic enzyme on the ethanol production, we first integrated E. coli maeB encoding NADP-dependent malic enzyme into the slr1176 site of the strain SYN007. Our results provided strong evidence that E. coli maeB was indeed shown to function as malic enzyme, catalyzing malate conversion to pyruvate. The improved ethanol production in the SYN009 strain may result from an increase in the intracellular level of the precursor pyruvate, which is subsequently metabolized for ethanol production. Equally important, the overexpression of this malic enzyme maeB led to not only the reversible oxidative decarboxylation of malate to pyruvate and CO 2 , but also accompanied with reduction of NADP + to NADPH [26]. Impressively, NADPH is an important reduced co-factor in cyanobacterial cells, and provides more favorable advantages for the NADPH-dependent metabolic pathways. Increasing NADPH production in cyanobacteria can further improve the production of the desired chemicals [27]. With respect to the final step of ethanol synthesis, the reduced co-factor NADPH may contribute to enhancing the catalytic activity of NADPH-dependent enzyme YqhD, which catalyze acetaldehyde to ethanol. The utilization of NADPH-dependent enzymes linking an exogenous biosynthetic pathway to the modulation of the cellular metabolism are of particular interest because they likely contribute to exploiting cyanobacterial NADPH pool in the biological processes of the ethanol production. Therefore, this synergy between NADPH-producing pathway and NADPH-consuming pathway may effectively improve the activities of NADP-dependent maeB and NADPH-dependent yqhD , which cause more metabolic carbon flux towards ethanol production. In the present study, all the engineered strains were cultivated under autotrophic conditions without optimizing growth medium, and faced many challenges in the ethanol yield. Traditionally, appropriate nutritional conditions such as certain amount of CO 2 or the reduced cofactor (NADPH) are needed to boost ethanol production. As shown in Fig. 5B, the synthetic ethanol of the final strain SY009 showed a dramatic increase within the first 4 days of cultivation, which almost linearly increased with the time, and then its production slightly increased. When the time consumption was counted for the whole process, the strain SYN009 showed relatively lower ethanol productivity of 93 mg L -1 day -1 after 14 days compared to other studies (Supplementary materials Fig S2). This may be the exhausted nutrient in the medium and the insufficient CO 2 in the air (less than 0.03% vol/vol) during the later stage of cultivation, which limits the cell growth rate. Thus, the requirement of high cell density became the critical factor controlling the ethanol yield in cyanobacteria. By pumping 5% CO 2 -air (vol/vol) into the photo-bioreactor, the ethanol yield of 5.5 g L -1 was achieved with a high cell density (OD 730 ≈15) after 26 days of fermentation [7]. In comparison, without using photo-bioreactor and pumping CO 2 , the strain SYN009 produced only 1.3 g L -1 of ethanol when the cell density was much lower, which OD 730 value was 1.37 after 14 days of culture. However, if the cell density were considered, the ethanol-producing efficiency of the strain SYN009 reached up to 68 mg OD 730 unit -1 L -1 day -1 , which was significantly better than that of the previous reports [7, 12, 14]. Thus, it will be one of important challenges for the engineered cyanobacteria to improve the ethanol productivity at high cell density over a long period. If overcame this problem, the utilization of the final strain SYN009 as photosynthetic biosystem would be expected to make the ethanol production more competitive in the future. Conclusions In this study, we genetically engineered a cyanobacterial strain that is entitled to converting atmospheric CO 2 into ethanol at high efficiency via stepwise genetic engineering. Combined with biosynthetic pathway bottleneck disruption and genetic interventions, the engineered strain showed a higher ethanol-producing efficiency (248 mg L -1 day -1 ) compared to previous studies under photoautotrophic conditions with nutrient limitations. Our findings indicated that the SYN009 strain ( ∆slr0301/pdc-yqhD , ∆slr1176/maeB ) would become a useful biosystem for photosynthetic production of ethanol through optimizing the fermentation conditions, and for expanding our knowledge of exploiting cyanobacteria to produce value chemicals directly from atmospheric CO 2 . Materials Strains and growth conditions The plasmids were constructed by using E. coli DH5α. The E. coli strains carrying the different plasmids were grown in the liquid LB medium or on agar plate containing corresponding antimicrobial agents, such as 50μl ml -1 spectinomycin (Sp R ), 50μl/ml kanamycin (Km R ), and 25μl ml -1 chloramphenicol (Cm R ). The wild-type and engineered Synechocystis 6803 strains were grown in the liquid BG-11 medium or on agar plates containing 1.5% agar, and kept at 30℃ under continuous illumination with an intensity of 50 μmol photons m -2 s -1 , unless otherwise noted. Engineered Synechocystis 6803 strains were grown in BG-11 medium supplemented with the corresponding antimicrobial agents according to details of each cyanobacterial strains. Plasmid Constructions The pMD18-T simple vector (Sangon Biotech) was used as a foundation to construct the plasmids, which were listed in Supplementary materials Table S1. Using PCR to amplify the fragments, the fragments and the vectors are double-digested by recombinase (NEW ENGLAND BioLabs Beijing, China) and ligated by T4 ligase (NEW ENGLAND BioLabs Beijing, China). All the primers were listed in Supplementary materials Table S2. For construction of gene deletion vector, plasmid pBE406 was constructed by inserting the slr0168 gene knockout cassette into the pMD18-T vector. The slr0168 gene knockout cassette was constructed by integrating 600 bp sequence located immediately upstream slr0168 (0168 up), spectinomycin resistance gene (Sp R ), and 600 bp sequence located immediately downstream slr0168 (0168 down). The upstream and downstream of the slr0168 gene were amplified by PCR from Synechocystis 6803 genome. The Sp R sequence was optimized and synthesized by Sangon Biotech as the template. Similar to the method of constructing pBE406, pMD-slr0301-Ω and pMD-slr1176-Ω were constructed and the specific method was shown in Supplementary materials Table S1. The Cm R sequence was also optimized and synthesized by Sangon Biotech. For construction of ethanol production vector, plasmid pBE01 was constructed by inserting PpetE-pdc-yqhD expression cassette and TrbcL terminator into the plasmid pBE406. The fragment sequence of PpetE and TrbcL were both PCR-amplified from Synechocystis 6803 genome. The pdc and yqhD gene was PCR-amplified from the nucleotide sequence that has been optimized and synthesized by Sangon Biotech. Plasmid pBE02 was constructed by replacing the promoter PpetE in pBE01 with PpsbA2s . Promoter PpsbA2s was also PCR-amplified from Synechocystis 6803 genome. Plasmid pBE03 was constructed by inserting PpsbA2s-pdc-yqhD expression cassette and TrbcL terminator into the plasmid pMD-slr0301-Ω . For construction of genetic intervention vector, plasmid pBE09 was constructed by inserting PpsbA2s -maeB expression cassette and TrbcL terminator into the pMD-slr1176-Ω vector. The maeB gene was amplified by PCR from E. coli genome. Construction of engineered strains . The constructed plasmids were independently transformed into Synechocystis 6803 according to the approach performed by previous studies [7]. Briefly, exponentially growing Synechocystis cultures were collected, washed with fresh BG11 medium three times, and then mixed with the corresponding plasmids. The mixture was incubated at the temperature of 30 °C for 5 hours under constant illumination and shaken periodically. The mixture was then streaked on a sterile filter membrane placed on BG11 solid medium and maintained at 30°C under continuous illumination. After 24 hours, the filter membrane was transferred to solid BG11 medium with the corresponding antibiotic. Single colonies appeared after two weeks of cultivation. A single transformant was sub-cultured stepwise on new BG11 plates containing increasing concentration of antibiotic for the occurrence of segregation and cultivated in a liquid medium for analysis. All the strains referred in this study were listed in Table 1. Analytical methods Synechocystis growth status was recorded by measuring transmittance at wavelength λ = 730 nm (OD730) using a spectrometer (YOKE INSTRUMENT L6 UV-Vis, Shanghai, China). The biomass is determined according to the published procedure [28]. OD730 was converted to biomass (dry cell weight g/L) by multiplying it by 0.177, which had been calculated from a calibration curve. HPLC analysis For the ethanol production assay, all the mutants were cultured in a fresh BG11 medium with initial OD730 = 0.1 and cultivated photoautotrophically in a flask. The BG11 medium of SYN001 contains 500 nM copper ions to induce the expression of ethanol-producing genes [29]. To get the samples, a certain amount of mutant cultures was regularly obtained and centrifuged at 10000×g for 2 minutes (MRK MG1450). Special filter membrane with 0.22- micron (Sigma - Aldrich) was used to filter the supernatant obtained by centrifugation. The filtered solution was used for ethanol analysis by the method of high-performance liquid chromatography (HPLC) [30]. According to the detection of HPLC, no ethanol was detected in the lysed precipitate. RT-qPCR The wild-type and ethanol-producing Synechocystis cultures (30 mL, OD 730 ≈0.6) was collected by centrifugation at 3500×g for 15min at 4℃ (BIORIDGE TGL-16M, Shanghai, China). Three biological replicates were carried out for each sample. RNA extraction and RT-qPCR analysis were performed according to the methods described previously [7]. The relative expression level of the targeted mRNA could be estimated using the calculation method of 2 -△△CT ; the higher the △CT value is, the less expression level of the targeted mRNA [31]. The endogenous gene 16S was selected as an internal reference gene. Declarations Acknowledgments We thank Prof. Degang Ning (Institute of Hydrobiology, Chinese Academy of Sciences) for the Synechocystis sp. PCC6803 wild-type strain and his technical expertise, Prof. Yangchun Yong (Biofuels Institute, School of Environment, Jiangsu University) for the HPLC analysis. Authors’ contributions EBG, HQ and JW designed the experiment. EBG and PY performed the experiments and collected the data. EBG, PY and ZZ analyzed the data and drafted the article. EBG, PY and HC critically revised the article. All authors approved the final draft for submission. Funding This work was supported by the National Natural Science Foundation of China (no. 31200019), the Open Fund of Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Sciences (LMB131001), Ningbo Clinical Research Center for Children’s Health and Diseases (2019A21002), Ningbo Science and Technology Innovation Team program (2014B82003), and the Open Funding Project of the State Key Laboratory of Biochemical Engineering, China (2018KF-02). Availability of data and material All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate Not applicable Consent for publication All authors consented to the publication of this work. Competing interests The authors declare that they have no competing interests. Author details 1 School of the Environment and Safety Engineering, Jiangsu University, No. 301, Xuefu Road, Zhenjiang City 212013, Jiangsu Province, China. 2 Ningbo Women and Children’s Hospital, Ningbo, China. 3 School of Life Sciences, Jiangsu University, No. 301, Xuefu Road, Zhenjiang City 212013, Jiangsu Province, China. References Parmar A, Singh NK, Pandey A, Gnansounou E, Madamwar D. Cyanobacteria and microalgae: a positive prospect for biofuels. Bioresour Technol. 2011; 102: 10163-10172. Rosgaard L, de Porcellinis AJ, Jacobsen JH, Frigaard NU, Sakuragi Y. Bioengineering of carbon fixation, biofuels, and biochemicals in cyanobacteria and plants. J Biotechnol. 2012; 162: 134-147. Zhou J, Zhu T, Cai Z, Li Y. From cyanochemicals to cyanofactories: a review and perspective. Microb Cell 2016;15: 2. Heidorn T, Camsund D, Huang HH, Lindberg P, Oliveira P, Stensjö K, Lindblad P. Synthetic biology in cyanobacteria engineering and analyzing novel functions. 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Gao EB, Kyere-Yeboah K, Wu J, Qiu H: Photoautotrophic production of p-coumaric acid using genetically engineered Synechocystis Pasteur Culture collection 6803. Algal Res. 2021; 54: 102180. Deng MD, Coleman JR. Ethanol synthesis by genetic engineering in cyanobacteria. Energy Environ Sci. 1999; 2: 523-528. Dexter J, Fu PC. Metabolic engineering of cyanobacteria for ethanol production. Energy Environ Sci. 2009; 2: 857-864. Velmurugan R, Incharoensakdi A. Co-cultivation of two engineered strains of Synechocystis PCC6803 results in improved ethanol production. Renew Energ. 2020; 146: 1124-1133. Liang F, Englund E, Lindberg P, Lindblad P. Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio. Metab Eng. 2018; 46: 51-59. Camsund D, Heidorn T, Lindblad P. Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium. J Biol Eng. 2014; 8: 4. Diao J, Song X, Zhang L, Cui J, Chen L, Zhang W. Tailoring cyanobacteria as a new platform for highly efficient synthesis of astaxanthin. Metab Eng. 2020; 61: 275-287. Zhang S, Bryan, DA. The tricarboxylic acid cycle in cyanobacteria. Science. 2011; 334: 1551-1553. Oliver JW, Machado IM, Yoneda H, Atsumi S. Cyanobacterial conversion of carbon dioxide to 2,3-butanediol. Proc Natl Acad Sci USA. 2013; 110: 1249-1254. van der Woude AD, Angermayr SA, Puthan VV, Osnato A, Hellingwerf KJ. Carbon sink removal: Increased photosynthetic production of lactic acid by Synechocystis sp. PCC6803 in a glycogen storage mutant. J Biotechnol. 2014; 184: 100-102. Gründel M, Scheunemann R, Lockau W, Zilliges Y. Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis PCC 6803. Microbiology. 2012; 158: 3032-3043. Namakoshi K, Nakajima T, Yoshikawa K, Toya Y, Shimizu H. Combinatorial deletions of glgC and phaCE enhance ethanol production in Synechocystis PCC 6803. J Biotechnol. 2016; 239: 13-19. Angermayr SA, van der Woude A.D, Correddu D, Vreugdenhil A, Verrone V, Hellingwerf K. Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis PCC6803. Biotechnol Biofuels. 2014; 1: 2-31. Young JD, Shastri AA, Stephanopoulos G, Morgan JA. Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis. Metab Eng. 2011; 13: 656-665. Akram M. Citric acid cycle and role of its intermediates in metabolism. Cell Biochem Biophys. 2014; 68: 475-478. Yoshikawa K, Hirasawa T, Shimizu H. Effect of malic enzyme on ethanol production by Synechocystis PCC 6803. J Biosci Bioeng. 2015; 119: 82-84. Huergo LF, Araújo GAT, Santos ASR, Gerhardt ECM, Pedrosa FO, Souza EM, Forchhammer K. The NADP-dependent malic enzyme MaeB is a central metabolic hub controlled by the acetyl-CoA to CoASH ratio. Biochim Biophys Acta Proteins Proteom. 2020; 1868: 140462. Choi YN, Park JM. Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis PCC 6803. Bioresour Technol. 2016; 213: 54-57. Brey LF, Włodarczyk AJ, Bang Thøfner JF, Burow M, Crocoll C, Nielsen I, Zygadlo Nielsen AJ, Jensen PE. Metabolic engineering of Synechocystis PCC 6803 for the production of aromatic amino acids and derived phenylpropanoids. Metab Eng. 2020; 57: 129-139. Ghassemian M, Wong B, Ferreira F, Markley JL, Straus NA. Cloning, sequencing and transcriptional studies of the genes for cytochrome c-553 and plastocyanin from Anabaena PCC 7120. Microbiology. 1996; 140: 1151-1159. Seo SO, Wang Y, Lu T, Jin YS, Blaschek HP. Characterization of a Clostridium beijerinckii spo0A mutant and its application for butyl butyrate production. Biotechnol Bioeng. 2017; 114: 106-112. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods. 2001; 25: 402-408. Tables Table 1 The strains constructed in this experiment Supplementary Files Supplementalmaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-193538","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":10218278,"identity":"a1a82568-9512-4b34-8cd4-5d208984b369","order_by":0,"name":"E-Bin Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYJCCDwwGNgyMDUAWD5E6GGcwGKSRrIXhMIRJlBZ598MHGz4UnE9s7j/A+OBtG4O8OSEthmfSEhtnGNwGEgnMhnPbGAx3NhDS0pBj/pgHrIWBTZq3jSHB4AAhLf1vDJv/GJxLbOw/wP6bKC3yEjmGzQwGBxIbGxLYmInSYiDxLLGxxyDZuHFGYrPknHMShhsI2tKffLDhxx872Y39hw9+eFNmI0/YFpgCwwZwZEoQUA+ypQHGIKx2FIyCUTAKRioAAMh6Q80l6z5UAAAAAElFTkSuQmCC","orcid":"","institution":"Jiangsu University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"E-Bin","middleName":"","lastName":"Gao","suffix":""},{"id":10218279,"identity":"d08d638a-af7c-4f01-a929-576f3494d482","order_by":1,"name":"Penglin Ye","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Penglin","middleName":"","lastName":"Ye","suffix":""},{"id":10218280,"identity":"f4bd7209-5a68-449d-bcd5-550a16c3f629","order_by":2,"name":"Haiyan Qiu","email":"","orcid":"","institution":"Ningbo Women and Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Qiu","suffix":""},{"id":10218281,"identity":"8c305d53-5c4b-4c6b-9b90-337af3e77826","order_by":3,"name":"Junhua Wu","email":"","orcid":"","institution":"Ningbo Women and Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junhua","middleName":"","lastName":"Wu","suffix":""},{"id":10218282,"identity":"7131e0cb-b4a4-444c-ae7a-1559d447a088","order_by":4,"name":"Huayou Chen","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huayou","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2021-01-31 10:50:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-193538/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-193538/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5977752,"identity":"fd298eea-df94-4660-818e-7c4df159fed5","added_by":"auto","created_at":"2021-02-15 17:10:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91973,"visible":true,"origin":"","legend":"Engineered biosynthetic pathway of bioethanol from carbon dioxide and sunlight in autotrophic Synechocystis sp. PCC6803. Native central carbon pathways are shown in blue arrows. The pyruvate-dependent ethanol pathway is shown in red arrows. Heterogeneous expression of malic enzyme (maeB) (marked by red arrow) conferred the conversion of malate to pyruvate. PEP pathway from pyruvate and glycogen biosynthesis pathway were blocked by knocking out the ppsA and glgC (marked by red crosses), respectively. ppsA, phosphoenolpyruvate synthase; glgC, glgA1 and glgA2, glycogen synthase; maeB, NADP-dependent malic enzyme from E. coli; me, malic enzyme from Synechocystis sp. PCC6803; pdc, pyruvate decarboxylase from Z. mobilis; yqhD, NADPH-dependent aldehyde reductase from E. coli; TCA, tricarboxylic acid cycle; CBB, Calvin-Benson-Bassham; RuBP, ribulose-1,5-bisphosphate; G1P, glucose-1-phosphate; ADP-Glu, ADP-Glucose; GAP, glyceraldehyde-3-phosphate; 3-PGA, 3-phosphoglycerate; 2PGA, 2-phosphoglyceric acid; PEP, phosphoenolpyruvate; MAL, malate; CIT, citrate; SUC, succinate.\n\n","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-193538/v1/685411e648a3a6423027a1f7.png"},{"id":5978123,"identity":"0bbbdff8-678c-4d7c-97ca-71b41049c019","added_by":"auto","created_at":"2021-02-15 17:13:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110784,"visible":true,"origin":"","legend":"Construction and expression of ethanol biosynthetic pathway in the engineered Synechocystis strains. \nA, Plasmid map of pBE01 and pBE02, respectively, used to transform Synechocystis sp. PCC 6803. B, PCR analysis of the insertion of ethanol biosynthetic pathway in the engineered strain SYN001 and SYN002, respectively. M-DNA marker, a-upstream of the slr0168 gene, b-Spectinomycin resistance gene, c-promoter PpetE (lift) or PpbsA2s (right), d-pdc gene, e-yqhD gene, f-Synechocystis TrbcL terminator, g-downstream of the slr0168 gene. C, Transcription levels of pdc and yqhD in the engineered strain SYN001 and SYN002, respectively. The endogenous gene 16S was selected as an internal reference gene. Error bars represent standard deviations of three biological replicates.\n","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-193538/v1/ff7e0da985c57e136b865a1e.png"},{"id":5977753,"identity":"3691f065-dddc-46ab-90a3-7eea8cb435a2","added_by":"auto","created_at":"2021-02-15 17:10:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18178,"visible":true,"origin":"","legend":"The detection and production of ethanol in the engineered Synechocystis strains.\nA, HPLC counts versus retention time is shown for the detection of ethanol in different strains. B, Ethanol production in different engineered strains. Error bars represent standard deviations of three biological replicates.\n","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-193538/v1/e84c9916e95166c7eafc14d5.png"},{"id":5977432,"identity":"9f0a99bc-97ff-4686-8046-6999fa6cdc48","added_by":"auto","created_at":"2021-02-15 17:07:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9200,"visible":true,"origin":"","legend":"The growth of Synechocystis sp. Wild-type (magenta), SYN003 (black), SYN004 (dark yellow), SYN007 (blue) under the autotrophic conditions. Each data point represents the mean and standard deviation of three independent experiments. ","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-193538/v1/18d0d4ba4f4ea824ffd603f9.png"},{"id":5977755,"identity":"18f11fc0-9b8a-490c-a168-b54e661672c5","added_by":"auto","created_at":"2021-02-15 17:10:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17770,"visible":true,"origin":"","legend":"Growth (A) and ethanol production (B) of Synechocystis SYN009 (pink circles) and wild-type (blue circles) under photoautotrophic condition. Error bars represent standard deviations of three biological replicates.\n\n","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-193538/v1/0e2bbe0391d151fc14a310bc.png"},{"id":15670824,"identity":"cb7087d7-1a94-4235-8319-1b1879fc25e5","added_by":"auto","created_at":"2021-11-18 14:02:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6719513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-193538/v1/77dc33ad-820f-48a7-a2ad-030cd1d6bb2d.pdf"},{"id":5977756,"identity":"debf3ab9-4a50-40c1-8b97-3540f90dfb7d","added_by":"auto","created_at":"2021-02-15 17:10:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":151659,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-193538/v1/5465c58b8925f81375d59e3f.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEnhancing Bioethanol Production by Deleting Phosphoenolpyruvate Synthase and ADP-Glucose Pyrophosphorylase, and Shunting Tricarboxylic Acid Cycle In \u003cem\u003eSynechocystis\u003c/em\u003e Sp. PCC 6803\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eDue to the high ability of directly integrating atmospheric CO\u003csub\u003e2\u003c/sub\u003e with sunlight into biomass, cyanobacteria have been increasingly proposed as one of the most promising biosystems for the solution to the global warming crises and the world energy growth [1]. Cyanobacteria are autotrophic prokaryotes that perform similar photosynthesis as higher plants [2]. However, compared to the traditional plants, cyanobacteria possess several advantages, including the simple inorganic nutrient requirement, the tolerant growth on non-arable land, and powerful genetic manipulation [3], which render photosynthetic cyanobacteria as attractive organisms for the direct production of important chemicals from atmospheric CO\u003csub\u003e2\u003c/sub\u003e through metabolic engineering methods [4]. Over the years, an enormous amount of the engineered cyanobacteria has been successfully developed to produce various industrial relevant chemicals, such as ethylene [5], isoprene [6], ethanol [7], isobutanol [8], acetone [9], and \u003cem\u003ep\u003c/em\u003e-coumaric acid [10].\u003c/p\u003e\n\u003cp\u003eEthanol is one of the major renewable biofuels that are a worldwide focus of main concern. Commercially, ethanol production is mostly based on the fermentation of starch or agricultural crops as feedstock [7]. The over-exploitation of this food-based raw material presents a significant bottleneck for expanding ethanol production. To avoid competition with the world food supply or agricultural land, the cyanobacteria-based method for ethanol production has been increasingly regarded as alternatives to biomass-based fermentation. In recent years, a lot of efforts have been made to realize ethanol production by engineered cyanobacteria. The first production of ethanol was successfully achieved in \u003cem\u003eSynechococcus elongatus\u003c/em\u003e PCC 7942 by expressing pyruvate decarboxylase (PDC) and alcohol dehydrogenase II (ADH II) from\u003cem\u003e Z. mobilis \u003c/em\u003e[11]. Soon afterward, \u003cem\u003eSynechocystis sp.\u003c/em\u003e PCC 6803 (hereafter \u003cem\u003eSynechocystis \u003c/em\u003e6803) was genetically engineered by expressing these two enzymes to produce ethanol, and obtained double the ethanol yield compared to \u003cem\u003eS. elongatus\u003c/em\u003e PCC7942 strain [12]. To further improve ethanol production, many desirable strategies were adopted to optimize abiotic and biotic factors that have effects on cyanobacterial cell growth and metabolisms. For example, overexpressing the ethanol-producing steps and blocking the production of storage polymers (glycogen and PHB) were performed in \u003cem\u003eSynechocystis\u003c/em\u003e 6803 to increase ethanol production [13]. Recently, another example has been significantly shown to improve the production of ethanol by engineered cyanobacteria with enhanced cell growth through overexpressing the Calvin-Benson-Bassham (CBB) cycle enzymes [14]. However, genetically engineered cyanobacteria-based biosystems still faced many challenges for ethanol production applications, such as redirecting carbon flux to the desired product instead of cellular biomass, promoting cell growth, and balancing the co-factor levels due to metabolic consumption of an introduced biosynthetic pathway. To enhance the ethanol production by cyanobacteria, more efficient and rational biosynthetic pathways need to be established through metabolic engineering.\u003c/p\u003e\n\u003cp\u003eIn this study, considerable attention is paid to achieve the ethanol production in the model cyanobacterium \u003cem\u003eSynechocystis\u003c/em\u003e 6803 by genetically integrating the genes encoding \u003cem\u003eZ. mobilis\u003c/em\u003e PDC and \u003cem\u003eE. coli\u003c/em\u003e YqhD into the\u003cem\u003e Synechocystis\u003c/em\u003e 6803 chromosome. To investigate the potential of optimizing metabolic pathways for improving ethanol productivity, the engineered cyanobacterial strains were genetically modified in a stepwise approach via inhibiting phosphoenolpyruvate pathway from pyruvate, removing glycogen storage, and shunting carbon metabolic flux of the tricarboxylic acid cycle. These approaches led to the high-efficient ethanol production directly from solar energy and atmospheric CO\u003csub\u003e2 \u003c/sub\u003eunder photoautotrophic conditions, and significantly contribute to enhancing the biological synthesis of the desired carbon-based biofuels.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eDetermining the metabolic pathways for the ethanol production\u003c/h2\u003e\n\u003cp\u003eTo achieve photosynthetic production of ethanol in \u003cem\u003eSynechocystis\u003c/em\u003e 6803, it is essential to rationally construct and optimize an exogenous biosynthetic pathway for redirecting carbon fluxes towards ethanol. Thus, the precursor intracellular level and the catalytic pathway efficiency are generally considered as two regulating factors for high-efficient production of ethanol. In cyanobacteria, the pyruvate and acetyl-CoA are two important intermediate metabolites, which are naturally accumulated in cells, and successfully exploited by several studies as the starting precursors to be converted into ethanol and other products. To evaluate which intermediate precursor is more efficient for ethanol production, we measured the intracellular content of the primary metabolites in the wild-type \u003cem\u003eSynechocystis\u003c/em\u003e 6803 strain. As shown in Fig. S1, the intracellular concentration of pyruvate reached up to an average of 1.05 \u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e (dry cell weight), which was approximately 3 times that of acetyl-CoA. This result indicated that \u003cem\u003eSynechocystis\u003c/em\u003e 6803 possesses a high potential ability to produce ethanol by using pyruvate as the starting precursor rather than acetyl-CoA.\u003c/p\u003e\n\u003cp\u003eThe major pathway of ethanol production was constructed by expressing two enzymes, \u003cem\u003eZ. mobilis \u003c/em\u003ePDC and\u003cem\u003e E. coli \u003c/em\u003eYqhD\u003cem\u003e. \u003c/em\u003eAs shown in Fig. 1, the pyruvate was directly converted to acetaldehyde and CO\u003csub\u003e2\u003c/sub\u003e by PDC, and subsequently, acetaldehyde was reduced by YqhD to generate ethanol and NADP\u003csup\u003e+\u003c/sup\u003e. The Cu\u003csup\u003e2+\u003c/sup\u003e inducible promoter \u003cem\u003ePpetE\u003c/em\u003e was initially selected to drive the expression of the exogenous genes. The promoter \u003cem\u003ePpetE\u003c/em\u003e and the codon-optimized genes were integrated into the neutral site (\u003cem\u003eslr0168\u003c/em\u003e) of \u003cem\u003eSynechocystis\u003c/em\u003e 6803 genome to generate the initial strain SYN001 (\u003cem\u003e∆slr0168/pdc-yqhD, PpetE\u003c/em\u003e) (Fig. 2A, Fig.2B). The resulting transformants were maintained in liquid BG-11 medium with the addition of 50mg/L spectinomycin. HPLC analysis showed that ethanol accumulated up to a yield of 230 mg L\u003csup\u003e-1\u003c/sup\u003e (OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;0.64) in the strain SYN001 after 7 days of photoautotrophic growth (Fig. 3A, Fig. 3B). According to the published studies [15, 16], the strength of the promoter \u003cem\u003ePpetE\u003c/em\u003e was categorized as \u0026ldquo;medium\u0026rdquo;, which led to relatively low expression levels of the targeted genes. This enabled us to hypothesize that the mRNA expression levels of the\u003cem\u003e pdc \u003c/em\u003eand \u003cem\u003eyqhD \u003c/em\u003egene were potentially correlated with ethanol production yield. To overcome the potential effects of possible low expression of the ethanol biosynthesis genes, we substituted the \u0026lsquo;medium\u0026rsquo; promoter \u003cem\u003ePpetE\u003c/em\u003e with the light-sensitive and strong promoter \u003cem\u003ePpsbA2s \u003c/em\u003eto construct the second producer SYN002 (\u003cem\u003e∆slr0168/pdc-yqhD\u003c/em\u003e,\u003cem\u003e PpsbA2s\u003c/em\u003e) (Fig. 2A, Fig.2B). After 7 days of cultivation, HPLC analysis demonstrated that 474 mg L\u003csup\u003e-1\u003c/sup\u003e (OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;0.64) of ethanol was accumulated in the culture medium, which was 2-fold higher than that of strain SYN001. These results demonstrated that enhancing the gene expression was required in \u003cem\u003eSynechocystis\u003c/em\u003e for the high-efficient production of ethanol. This is also consistent with previous studies where the relatively high expression levels of biosynthetic genes were required to improve the production of the desired chemicals.\u003c/p\u003e\n\u003ch2\u003eEliminating \u003c/strong\u003e\u003cstrong\u003ephosphoenolpyruvate\u003c/strong\u003e\u003cstrong\u003e pathway from pyruvate\u003c/h2\u003e\n\u003cp\u003eDue to continuous production and consumption by several metabolic pathways, the intermediate pyruvate undergoes dynamic fluctuation at the intracellular level. This rendered us attempt to lessen the competitive consumption of the precursor pyruvate by inhibiting the catalytic activity of phosphoenolpyruvate synthase encoded by the \u003cem\u003eslr0301\u003c/em\u003e gene of \u003cem\u003eSynechocystis\u003c/em\u003e 6803 genome. Theoretically, disruption of the \u003cem\u003eslr0301\u003c/em\u003e gene would metabolically block the catalytic conversion of the pyruvate to phosphoenolpyruvate, and then could increase the endogenous carbon flux from pyruvate to ethanol. To test if the catalytic inhibition of phosphoenolpyruvate (PEP) synthase activity could contribute to ethanol formation, the engineered strain SYN003 (\u003cem\u003e∆slr0301/pdc-yqhD\u003c/em\u003e) was constructed by transforming the plasmid pBE03 inculcating the \u003cem\u003eslr0301 \u003c/em\u003eknockout cassette into the wide-type \u003cem\u003eSynechocystis\u003c/em\u003e strain. The ethanol-producing assay was performed to compare the yield of the newly engineered strain SYN003 with the strain SYN001 and SYN002. The ethanol yield was improved to 600 mg L\u003csup\u003e-1\u003c/sup\u003e (OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;0.64) after 7 days of photoautotrophic growth. The yield of the strain SYN003 was approximately 2.6-fold and 1.3-fold higher than that of SYN001 and SYN002, respectively. This result gave an implication that catalytic inactivation of phosphoenolpyruvate synthase led to the level increase of the precursor pyruvate, and then boosted the ethanol formation. To determine whether disruption of the \u003cem\u003eslr0301\u003c/em\u003e gene has negative effects on the photoautotrophic growth of the engineered\u003cem\u003e Synechocystis\u003c/em\u003e strain SYN003, the time-courses of OD\u003csub\u003e730\u003c/sub\u003e were performed to compare the growth pattern of the strain SYN003 and SYN004 (\u003cem\u003e∆slr0301\u003c/em\u003e) with the wild-type strains. Obviously, no differences were observed between the engineered strains and the wild-type strain (Fig. 4), indicating that the deletion of the \u003cem\u003eslr0301\u003c/em\u003e gene has no significant effects on the physiological activities of \u003cem\u003eSynechocystis \u003c/em\u003e6803. Based on these results, rational optimization of the pyruvate consumption pathway might provide a useful strategy that can contribute to effectively increase the ethanol biosynthesis in the engineered strain of \u003cem\u003eSynechocystis\u003c/em\u003e 6803.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eBlocking glycogen synthesis pathway\u003c/h2\u003e\n\u003cp\u003eTo enhance photosynthetic carbon flux towards chemical production by cyanobacteria, removing the glycogen biosynthetic pathway would become a helpful strategy for the generation of biofuels and chemicals. In this study, to test if inactivation of glycogen synthesis contributes to the ethanol production in the phosphoenolpyruvate synthase-deficient strain (\u003cem\u003e∆slr0301/pdc-yqhD\u003c/em\u003e), we also performed complete inhibition of the AGPase activity by disrupting the \u003cem\u003eslr1176\u003c/em\u003e gene of \u003cem\u003eSynechocystis\u003c/em\u003e 6803 genome. The engineered strain SYN007 (\u003cem\u003e∆slr0301/pdc-yqhD\u003c/em\u003e, \u003cem\u003e∆slr1176\u003c/em\u003e) was constructed by transforming the plasmid pMD-slr1176-\u0026Omega; inculcating the \u003cem\u003eslr1176 \u003c/em\u003egene knockout cassette into the strain SYN003. The ethanol yield in this double-knockout strain reached up to 689 mg L\u003csup\u003e-1\u003c/sup\u003e (OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;0.57) under the photoautotrophic conditions. The strain SYN007 showed about 1.5-fold ethanol yield compared with the starting strain SYN002. Similar to the strain SYN003 and SYN004, no remarkable differences were shown between the engineered SYN007 and the wild-type strain (Fig. 4). These data confirmed that complete inactivation of glycogen synthesis can be beneficial for the direct conversion of the fixed carbon in photosynthesis to the desired ethanol. This is also consistent with the previous studies that blocking the glycogen synthesis pathway can shift excess carbon from the glycolytic pathway and pentose phosphate pathway to a favorable biosynthetic pathway.\u003c/p\u003e\n\u003ch2\u003eMetabolic interventions of the TCA cycle\u003c/h2\u003e\n\u003cp\u003eIt has been reported that the major portion of the fixed carbon in \u003cem\u003eSynechocystis\u003c/em\u003e 6803 was metabolically consumed by the aerobic TCA cycle, which is one of the central carbon metabolisms in cyanobacteria [17]. Those carbon reserve metabolites produced from the TCA cycle are of great interest because they are efficient substrates for the production of ethanol or other biofuels. Thus, in the next optimization step, we attempted to improve ethanol production by shunting the metabolic route of \u003cem\u003eSynechocystis\u003c/em\u003e TCA cycle metabolism. The metabolic intervention of the TCA cycle was conducted by over-expressing NADP-dependent malic enzyme (\u003cem\u003emae\u003c/em\u003eB) from \u003cem\u003eE. coli\u003c/em\u003e. The \u003cem\u003emaeB\u003c/em\u003e gene was introduced into the \u003cem\u003eslr1176\u003c/em\u003e site of the strain SYN003, establishing SYN009 (\u003cem\u003e∆slr0301/pdc-yqhD\u003c/em\u003e, \u003cem\u003e∆slr1176/maeB\u003c/em\u003e). After 7 days of photoautotrophic growth, the strain SYN009 was able to generate ethanol, which the production yield reached up to 1.09 g L\u003csup\u003e-1\u003c/sup\u003e (OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;0.7) (Fig. 3B), and showed more than 1.6-fold improved ethanol yield compared with the strain SYN007. To our surprise, under photoautotrophic conditions, the strain SYN009 displayed normal growth and propagated steadily over culture time, and showed 30% higher cell density than the wide-type strains (Fig. 5A).\u003c/p\u003e\n\u003cp\u003eTo indicate the hypothesis that the engineered strains produce higher yield of ethanol over long-term growth, the strain SYN009 was cultivated for up to 14 days. As expected, the ethanol accumulation was increased by 30% and improved to 1.3 g L\u003csup\u003e-1\u003c/sup\u003e (OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;1.37) at 14 days of photoautotrophic culture. Interestingly, the ethanol yield in strain SYN009 was increased dramatically during the growth stage of days 0-4. During each of these days, the ethanol productivity was greater than 200 mg L\u003csup\u003e-1\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e (Supplementary materials Fig. S2). The highest ethanol productivity reached 248 mg L\u003csup\u003e-1\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e (OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;0.70) at the 4th day. After day 4, the ethanol yield showed a slight increase, but the ethanol productivity significantly decreased to less than 100 mg/L/day. This may be that the insufficient supply of CO\u003csub\u003e2\u003c/sub\u003e and light limits ethanol production of the strain SYN009 under photoautotrophic conditions. Taken together, these data indicated that a basic photo-biosystem for ethanol production was developed in our experiment through metabolic engineering optimization, which contributes to redirecting the metabolic carbon flux towards ethanol\u0026nbsp;production.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt has been reported that the metabolic imbalance of endogenous metabolism and the biosynthetic pathway often limits the desired chemical productivity and yield by microbial systems [18]. To enhance the carbon flux towards ethanol production, it is necessary to counterbalance the contradictory relationship between endogenous metabolism and the biosynthetic pathways. Therefore, we focused on the genetically engineering of a cyanobacterial strain for enhancing the capacity of metabolic flux toward the pyruvate, an important intermediate metabolite for ethanol production. For this purpose, we developed several modified metabolic bypasses to boost the intracellular supply of pyruvate in the engineered \u003cem\u003eSynechocysits\u003c/em\u003e harboring the ethanol-producing pathway. Our integrative approach led to successful improvement of the ethanol yield and productivity via stepwise metabolic engineering, e.g., disruption of consuming pyruvate pathways and reorientation of the carbon flux of the TCA cycle towards pyruvate.\u003c/p\u003e\n\u003cp\u003eIn the glycolytic pathway, phosphoenolpyruvate (PEP) is catalyzed by pyruvate kinase into pyruvate, and reversibly, the pyruvate is converted into PEP through the activity of PEP synthase (PpsA). This means that PpsA could be an attractive target for pyruvate forming metabolic bypass from PEP. In \u003cem\u003eSynechocystis \u003c/em\u003e6803, PpsA is encoded by the \u003cem\u003eslr0301\u003c/em\u003egene. To the best of our knowledge, no experimental evidence has to date been reported on the potential effects of the disruption of \u003cem\u003eSynechocystis\u003c/em\u003e PpsA on the ethanol production. To test if this could be the case in \u003cem\u003eSynechocystis\u003c/em\u003e, we constructed the strain SYN003 by deleting the PEP synthase coding locus: \u003cem\u003eslr0301\u003c/em\u003e. As expected, with the inhibition of PEP synthase activity, the ethanol production yield in the strain SYN003 showed significant increase when compared to the strain SYN001 and SYN002. Moreover, the cell growth of the PEP synthase-deficient\u003cem\u003e Synechocystis \u003c/em\u003eis normal and similar to that of the wide-type strains. These results suggested that the optimized metabolic bypass can regulate the strain SYN003 intracellular metabolism to improve the pyruvate supply for ethanol production. It can be suggested that deletion of the PEP synthase contributed to the yield of ethanol production by repression of PEP synthesis.\u003c/p\u003e\n\u003cp\u003eTo further improve the ethanol yield in our study, an alternative strategy is to perform the complete inhibition of glycogen synthesis pathway. Glycogen is generated from CBB cycle, and considered as one of major storage components for carbon resources in cyanobacteria. Significantly, if \u003cem\u003eSynechocystis\u003c/em\u003e 6803 cells lack the ability of glycogen synthesis, they will show an overflow of carbon metabolism leading to the excretion of pyruvate [19]. This effect may be hijacked for product formation by introducing a pyruvate-utilizing reaction such as ethanol production [20]. Several studies have been conducted to increase the ethanol production in cyanobacteria by deleting the glycogen synthesis pathway [13, 21]. To check if this could also be the case for \u003cem\u003eSynechocystis\u003c/em\u003e, we optimized the strain SYN003 by knocking out the \u003cem\u003eglgC\u003c/em\u003e: \u003cem\u003eslr1176\u003c/em\u003e gene encoding AGPase to obtain the glycogen-deficient strain. As expected, the strain SYN007 produced more ethanol than the strain SYN003. The marked increase of ethanol yield in the strain SYN007 strongly confirmed that complete inhibition of glycogen synthesis could contribute to the ethanol production. Also, this is the first investigation of the effect of combinatorial inhibition of PEP synthase and glycogen synthesis on ethanol production. It can be hypothesized that the production of the increased level of ethanol in the stain SYN007 could be caused by the repression of the cell growth and glycogen storage. Although the enhancement of ethanol production was observed in the strain SYN007, it would be necessary to preform metabolomics and proteomics analysis for further understanding the regulating mechanisms or metabolic network.\u003c/p\u003e\n\u003cp\u003eIn this study, considerable research attention has been made to remove the potential bottlenecks in ethanol biosynthetic pathway. As the intercellular concentration of pyruvate, the major intermediate precursor, is metabolically controlled by several endogenous pathways. Traditionally, it holds the view that the pyruvate was mainly generated from the PEP through pyruvate kinase [7]. However, recent studies have shown that a carbon flux is significantly channeled via the TCA cycle through the malic enzyme to pyruvate, rather than generating pyruvate directly from the ATP-generating reaction catalyzed by pyruvate kinase [22, 23]. Thus, the metabolic interference of the TCA cycle is suggested as an alternative way to boost ethanol production [24]. For this purpose, a previous study has been performed in an attempt to investigate the effects of an endogenous gene \u003cem\u003eslr0721 \u003c/em\u003eencoding malic enzyme (\u003cem\u003eme\u003c/em\u003e) in \u003cem\u003eSynechocysitis\u003c/em\u003e 6803 on the ethanol production [25], suggesting that the ethanol production was associated with the optimal level of malic enzyme activity in \u003cem\u003eSynechocysitis\u003c/em\u003e 6803. To test whether the potential effects of exogenous malic enzyme on the ethanol production, we first integrated \u003cem\u003eE. coli maeB\u003c/em\u003e encoding NADP-dependent malic enzyme into the \u003cem\u003eslr1176\u003c/em\u003e site of the strain SYN007. Our results provided strong evidence that \u003cem\u003eE. coli maeB\u003c/em\u003e was indeed shown to function as malic enzyme, catalyzing malate conversion to pyruvate. The improved ethanol production in the SYN009 strain may result from an increase in the intracellular level of the precursor pyruvate, which is subsequently metabolized for ethanol production. Equally important, the overexpression of this malic enzyme \u003cem\u003emaeB\u003c/em\u003e led to not only the reversible oxidative decarboxylation of malate to pyruvate and CO\u003csub\u003e2\u003c/sub\u003e, but also accompanied with reduction of NADP\u003csup\u003e+\u003c/sup\u003e to NADPH [26]. Impressively, NADPH is an important reduced co-factor in cyanobacterial cells, and provides more favorable advantages for the NADPH-dependent metabolic pathways. Increasing NADPH production in cyanobacteria can further improve the production of the desired chemicals [27]. With respect to the final step of ethanol synthesis, the reduced co-factor NADPH may contribute to enhancing the catalytic activity of NADPH-dependent enzyme YqhD, which catalyze acetaldehyde to ethanol. The utilization of NADPH-dependent enzymes linking an exogenous biosynthetic pathway to the modulation of the cellular metabolism are of particular interest because they likely contribute to exploiting cyanobacterial NADPH pool in the biological processes of the ethanol production. Therefore, this synergy between NADPH-producing pathway and NADPH-consuming pathway may effectively improve the activities of NADP-dependent \u003cem\u003emaeB \u003c/em\u003eand NADPH-dependent \u003cem\u003eyqhD\u003c/em\u003e, which cause more metabolic carbon flux towards ethanol production.\u003c/p\u003e\n\u003cp\u003eIn the present study, all the engineered strains were cultivated under autotrophic conditions without optimizing growth medium, and faced many challenges in the ethanol yield. Traditionally, appropriate nutritional conditions such as certain amount of CO\u003csub\u003e2\u003c/sub\u003e or the reduced cofactor (NADPH) are needed to boost ethanol production. As shown in Fig. 5B, the synthetic ethanol of the final strain SY009 showed a dramatic increase within the first 4 days of cultivation, which almost linearly increased with the time, and then its production slightly increased. When the time consumption was counted for the whole process, the strain SYN009 showed relatively lower ethanol productivity of 93 mg L\u003csup\u003e-1\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e after 14 days compared to other studies (Supplementary materials Fig S2). This may be the exhausted nutrient in the medium and the insufficient CO\u003csub\u003e2\u003c/sub\u003e in the air (less than 0.03% vol/vol) during the later stage of cultivation, which limits the cell growth rate. Thus, the requirement of high cell density became the critical factor controlling the ethanol yield in cyanobacteria. By pumping 5% CO\u003csub\u003e2\u003c/sub\u003e-air (vol/vol) into the photo-bioreactor, the ethanol yield of 5.5 g L\u003csup\u003e-1\u003c/sup\u003e was achieved with a high cell density (OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;15) after 26 days of fermentation [7]. In comparison, without using photo-bioreactor and pumping CO\u003csub\u003e2\u003c/sub\u003e, the strain SYN009 produced only 1.3 g L\u003csup\u003e-1\u003c/sup\u003e of ethanol when the cell density was much lower, which OD\u003csub\u003e730\u003c/sub\u003e value was 1.37 after 14 days of culture. However, if the cell density were considered, the ethanol-producing efficiency of the strain SYN009 reached up to 68 mg OD\u003csub\u003e730\u003c/sub\u003e unit\u003csup\u003e-1\u003c/sup\u003e L\u003csup\u003e-1\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e, which was significantly better than that of the previous reports [7, 12, 14]. Thus, it will be one of important challenges for the engineered cyanobacteria to improve the ethanol productivity at high cell density over a long period. If overcame this problem, the utilization of the final strain SYN009 as photosynthetic biosystem would be expected to make the ethanol production more competitive in the future.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we genetically engineered a cyanobacterial strain that is entitled to converting atmospheric CO\u003csub\u003e2 \u003c/sub\u003einto ethanol at high efficiency via stepwise genetic engineering. Combined with biosynthetic pathway bottleneck disruption and genetic interventions, the engineered strain showed a higher ethanol-producing efficiency (248 mg L\u003csup\u003e-1\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e) compared to previous studies under photoautotrophic conditions with nutrient limitations. Our findings indicated that the SYN009 strain (\u003cem\u003e∆slr0301/pdc-yqhD\u003c/em\u003e, \u003cem\u003e∆slr1176/maeB\u003c/em\u003e) would become a useful biosystem for photosynthetic production of ethanol through optimizing the fermentation conditions, and for expanding our knowledge of exploiting cyanobacteria to produce value chemicals directly from atmospheric CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e"},{"header":"Materials ","content":"\u003ch2\u003eStrains and growth conditions\u003c/h2\u003e\n\u003cp\u003eThe plasmids were constructed by using \u003cem\u003eE. coli \u003c/em\u003eDH5\u0026alpha;. The \u003cem\u003eE. coli \u003c/em\u003estrains carrying the different plasmids were grown in the liquid LB medium or on agar plate containing corresponding antimicrobial agents, such as 50\u0026mu;l ml\u003csup\u003e-1\u003c/sup\u003e spectinomycin (Sp\u003csup\u003eR\u003c/sup\u003e), 50\u0026mu;l/ml kanamycin (Km\u003csup\u003eR\u003c/sup\u003e), and 25\u0026mu;l ml\u003csup\u003e-1\u003c/sup\u003e chloramphenicol (Cm\u003csup\u003eR\u003c/sup\u003e). The wild-type and engineered \u003cem\u003eSynechocystis\u003c/em\u003e 6803 strains were grown in the liquid BG-11 medium or on agar plates containing 1.5% agar, and kept at 30℃ under continuous illumination with an intensity of 50 \u0026mu;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, unless otherwise noted. Engineered \u003cem\u003eSynechocystis\u003c/em\u003e 6803 strains were grown in BG-11 medium supplemented with the corresponding antimicrobial agents according to details of each cyanobacterial strains.\u003c/p\u003e\n\u003ch2\u003ePlasmid Constructions\u003c/h2\u003e\n\u003cp\u003eThe pMD18-T simple vector (Sangon Biotech) was used as a foundation to construct the plasmids, which were listed in Supplementary materials Table S1. Using PCR to amplify the fragments, the fragments and the vectors are double-digested by recombinase (NEW ENGLAND BioLabs Beijing, China) and ligated by T4 ligase (NEW ENGLAND BioLabs Beijing, China). All the primers were listed in Supplementary materials Table S2.\u003c/p\u003e\n\u003cp\u003eFor construction of gene deletion vector, plasmid pBE406 was constructed by inserting the slr0168 gene knockout cassette into the pMD18-T vector. The slr0168 gene knockout cassette was constructed by integrating 600 bp sequence located immediately upstream slr0168 (0168 up), spectinomycin resistance gene (Sp\u003csup\u003eR\u003c/sup\u003e), and 600 bp sequence located immediately downstream slr0168 (0168 down). The upstream and downstream of the slr0168 gene were amplified by PCR from \u003cem\u003eSynechocystis \u003c/em\u003e6803 genome. The Sp\u003csup\u003eR\u003c/sup\u003e sequence was optimized and synthesized by Sangon Biotech as the template. Similar to the method of constructing pBE406, pMD-slr0301-\u0026Omega; and pMD-slr1176-\u0026Omega; were constructed and the specific method was shown in Supplementary materials Table S1. The Cm\u003csup\u003eR \u003c/sup\u003esequence was also optimized and synthesized by Sangon Biotech.\u003c/p\u003e\n\u003cp\u003eFor construction of ethanol production vector, plasmid pBE01 was constructed by inserting \u003cem\u003ePpetE-pdc-yqhD\u003c/em\u003e expression cassette and \u003cem\u003eTrbcL\u003c/em\u003e terminator into the plasmid pBE406. The fragment sequence of \u003cem\u003ePpetE\u003c/em\u003e and \u003cem\u003eTrbcL\u003c/em\u003e were both PCR-amplified from \u003cem\u003eSynechocystis\u003c/em\u003e 6803 genome. The \u003cem\u003epdc\u003c/em\u003e and \u003cem\u003eyqhD\u003c/em\u003e gene was PCR-amplified from the nucleotide sequence that has been optimized and synthesized by Sangon Biotech. Plasmid pBE02 was constructed by replacing the promoter \u003cem\u003ePpetE\u003c/em\u003e in pBE01 with \u003cem\u003ePpsbA2s\u003c/em\u003e\u003cem\u003e. \u003c/em\u003ePromoter \u003cem\u003ePpsbA2s\u003c/em\u003e was also PCR-amplified from \u003cem\u003eSynechocystis\u003c/em\u003e 6803 genome. Plasmid pBE03 was constructed by inserting \u003cem\u003ePpsbA2s-pdc-yqhD\u003c/em\u003e expression cassette and \u003cem\u003eTrbcL\u003c/em\u003e terminator into the plasmid pMD-slr0301-\u0026Omega; .\u003c/p\u003e\n\u003cp\u003eFor construction of genetic intervention vector, plasmid pBE09 was constructed by inserting \u003cem\u003ePpsbA2s\u003c/em\u003e\u003cem\u003e-maeB \u003c/em\u003eexpression cassette and \u003cem\u003eTrbcL\u003c/em\u003e terminator into the pMD-slr1176-\u0026Omega; vector. The \u003cem\u003emaeB\u003c/em\u003e gene was amplified by PCR from \u003cem\u003eE. coli\u003c/em\u003e genome.\u003c/p\u003e\n\u003ch2\u003eConstruction\u003c/strong\u003e\u003cstrong\u003e of engineered strains\u003cem\u003e.\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe constructed plasmids were independently transformed into \u003cem\u003eSynechocystis \u003c/em\u003e6803 according to the approach performed by previous studies [7]. Briefly, exponentially growing \u003cem\u003eSynechocystis \u003c/em\u003ecultures were collected, washed with fresh BG11 medium three times, and then mixed with the corresponding plasmids. The mixture was incubated at the temperature of 30 \u0026deg;C for 5 hours under constant illumination and shaken periodically. The mixture was then streaked on a sterile filter membrane placed on BG11 solid medium and maintained at 30\u0026deg;C under continuous illumination. After 24 hours, the filter membrane was transferred to solid BG11 medium with the corresponding antibiotic. Single colonies appeared after two weeks of cultivation. A single transformant was sub-cultured stepwise on new BG11 plates containing increasing concentration of antibiotic for the occurrence of segregation and cultivated in a liquid medium for analysis. All the strains referred in this study were listed in Table 1.\u003c/p\u003e\n\u003ch2\u003eAnalytical methods \u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eSynechocystis\u003c/em\u003e growth status was recorded by measuring transmittance at wavelength \u0026lambda; = 730 nm (OD730) using a spectrometer (YOKE INSTRUMENT L6 UV-Vis, Shanghai, China). The biomass is determined according to the published procedure [28]. OD730 was converted to biomass (dry cell weight g/L) by multiplying it by 0.177, which had been calculated from a calibration curve.\u003c/p\u003e\n\u003ch2\u003eHPLC analysis\u003c/h2\u003e\n\u003cp\u003eFor the ethanol production assay, all the mutants were cultured in a fresh BG11 medium with initial OD730 = 0.1 and cultivated photoautotrophically in a flask. The BG11 medium of SYN001 contains 500 nM copper ions to induce the expression of ethanol-producing genes [29]. To get the samples, a certain amount of mutant cultures was regularly obtained and centrifuged at 10000\u0026times;g for 2 minutes (MRK MG1450). Special filter membrane with 0.22- micron (Sigma - Aldrich) was used to filter the supernatant obtained by centrifugation. The filtered solution was used for ethanol analysis by the method of high-performance liquid chromatography (HPLC) [30]. According to the detection of HPLC, no ethanol was detected in the lysed precipitate.\u003c/p\u003e\n\u003ch2\u003eRT-qPCR \u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;The wild-type and ethanol-producing \u003cem\u003eSynechocystis\u003c/em\u003e cultures (30 mL, OD\u003csub\u003e730\u003c/sub\u003e\u0026asymp;0.6) was collected by centrifugation at 3500\u0026times;g for 15min at 4℃ (BIORIDGE TGL-16M, Shanghai, China). Three biological replicates were carried out for each sample. RNA extraction and RT-qPCR analysis were performed according to the methods described previously [7]. The relative expression level of the targeted mRNA could be estimated using the calculation method of 2\u003csup\u003e-△△CT\u003c/sup\u003e; the higher the △CT value is, the less expression level of the targeted mRNA [31]. The endogenous gene 16S was selected as an internal reference gene.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank Prof. Degang Ning (Institute of Hydrobiology, Chinese Academy of Sciences) for the \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC6803 wild-type strain and his technical expertise, Prof. Yangchun Yong (Biofuels Institute, School of Environment, Jiangsu University) for the HPLC analysis.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eEBG, HQ and JW designed the experiment. EBG and PY performed the experiments and collected the data. EBG, PY and ZZ analyzed the data and drafted the article. EBG, PY and HC critically revised the article. All authors approved the final draft for submission.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (no. 31200019), the Open Fund of Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Sciences (LMB131001), Ningbo Clinical Research Center for Children\u0026rsquo;s Health and Diseases (2019A21002), Ningbo Science and Technology Innovation Team program (2014B82003), and the Open Funding Project of the State Key Laboratory of Biochemical Engineering, China (2018KF-02).\u003c/p\u003e\n\u003ch2\u003eAvailability of data and material\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors consented to the publication of this work.\u003c/p\u003e\n\u003ch2\u003eCompeting interests \u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor details\u003c/h2\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eSchool of the Environment and Safety Engineering, Jiangsu University, No. 301, Xuefu Road, Zhenjiang City 212013, Jiangsu Province, China.\u003csup\u003e2\u003c/sup\u003eNingbo Women and Children\u0026rsquo;s Hospital, Ningbo, China. \u003csup\u003e3\u003c/sup\u003eSchool of Life Sciences, Jiangsu University, No. 301, Xuefu Road, Zhenjiang City 212013, Jiangsu Province, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eParmar A, Singh NK, Pandey A, Gnansounou E, Madamwar D. 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Methods. 2001; 25: 402-408.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e The strains constructed in this experiment\u003c/p\u003e\n\u003cp\u003e\u003cimg 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Tricarboxylic acid cycle","lastPublishedDoi":"10.21203/rs.3.rs-193538/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-193538/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe outstanding ability of directly assimilating carbon dioxide and sunlight to produce biofuels and chemicals impels photosynthetic cyanobacteria to become attractive organisms for the solution to the global warming crises and the world energy growth. The cyanobacteria-based method for ethanol production has been increasingly regarded as alternatives to food biomass-based fermentation and traditional petroleum-based production. Therefore, we engineered the model cyanobacterium \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 to synthesize ethanol and optimized the biosynthetic pathways for improving ethanol production under photoautotrophic conditions.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn this study, we successfully achieved the photosynthetic production of ethanol from atmospheric carbon dioxide by an engineered mutant \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 with over-expressing the heterologous genes encoding\u003cem\u003e Zymomonas mobilis\u003c/em\u003e pyruvate decarboxylase (PDC) and\u003cem\u003e Escherichia coli\u003c/em\u003e NADPH-dependent alcohol dehydrogenase (YqhD). The engineered strain was further optimized by an alternative engineering approach to improve cell growth, and increase the intracellular supply of the precursor pyruvate for ethanol production under photoautotrophic conditions. This approach includes blocking phosphoenolpyruvate synthetic pathway from pyruvate, removing glycogen storage, and shunting carbon metabolic flux of tricarboxylic acid cycle. Through redirecting and optimizing the\u0026nbsp;metabolic carbon flux of\u003cem\u003e Synechocystis\u003c/em\u003e, a high ethanol-producing efficiency was achieved (248 mg L\u003csup\u003e-1\u003c/sup\u003e day\u003csup\u003e-1\u003c/sup\u003e) under photoautotrophic conditions with atmospheric CO\u003csub\u003e2\u003c/sub\u003e as the sole carbon source. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe engineered strain SYN009 (\u003cem\u003e∆slr0301/pdc-yqhD\u003c/em\u003e, \u003cem\u003e∆slr1176/maeB\u003c/em\u003e) would become a valuable biosystem for photosynthetic production of ethanol and for expanding our knowledge of exploiting cyanobacteria to produce value chemicals directly from atmospheric CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","manuscriptTitle":"Enhancing Bioethanol Production by Deleting Phosphoenolpyruvate Synthase and ADP-Glucose Pyrophosphorylase, and Shunting Tricarboxylic Acid Cycle In Synechocystis Sp. PCC 6803","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-15 17:07:48","doi":"10.21203/rs.3.rs-193538/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3a4f9d1a-0fd0-4fa6-8e67-06344047648b","owner":[],"postedDate":"February 15th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2411041,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-06-10T16:21:18+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-15 17:07:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-193538","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-193538","identity":"rs-193538","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}