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Sep 20, 2018 - Synthetic biology emerges as a powerful approach for unlocking the potential of cyanobacteria to produce various chemicals. However, th...
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Enhancing Light-Driven 1,3-Propanediol Production by Using Natural Compartmentalization of Differentiated Cells Hongyu Liu, Jun Ni, Ping Xu, and Fei Tao* State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China

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ABSTRACT: Synthetic biology emerges as a powerful approach for unlocking the potential of cyanobacteria to produce various chemicals. However, the highly oxidative intracellular environment of cyanobacteria is incompatible to numerous introduced enzymes from anaerobes. In this study, we explore a strategy based on natural compartmentalization of cyanobacterial heterocysts to overcome the incompatibility. Hence, the oxygen-sensitive 1,3-propanediol (1,3PDO) biosynthetic pathway was selected as a model and insulated in heterocysts to evaluate the proposed strategy. Thus, the genes from different sources for 1,3-PDO production were tandemly arrayed with promoter, resulting the assembled 1,3-PDO synthetic cassettes. Then the synthetic cassettes were integrated into the chromosome of Anabaena sp. strain PCC7120 by homologous recombination, respectively. The engineered strain P11 containing the genes from facultative anaerobe Klebsiella pneumoniae (cassette KP) accumulated 46.0 mg L−1 of 1,3-PDO when heterocysts were present, which is approximately 1.7-fold higher than that of no heterocysts. As for the strains (P12, P13, and P14) containing the genes from strictly anaerobic bacterium Clostridium butyricum (cassette CB), the product 1,3-PDO could only be detected when heterocysts were present. These results indicate that insulation of the oxygen-sensitive 1,3-PDO pathway with heterocysts is an effective way to protect these enzymes in cyanobacteria. The strategy may have the potential of serving as a universal strategy to overcome the incompatibility of oxygen-sensitivity in synthetic biology. KEYWORDS: incompatibility, cyanobacterium, oxygen-sensitivity, compartmentalization, 1,3-propanediol, heterocyst

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pathways and cyanobacteria. There are many reasons for the incompatibility such as oxygen-sensitivity, cofactor bias, and differences in tolerance to metabolites.17 It is considered that the highly oxidative intracellular environment of cyanobacteria is toxic to various oxygen-sensitive enzymes and contributes to one of the major incompatibility of introduced heterologous pathways in cyanobacteria.17,18 To bypass the obstacle of oxygen-sensitivity, many studies have focused on substituting an oxygen-sensitive enzyme with an oxygen-tolerant one, optimizing cultivation conditions or constructing coculture system.8,19,20 It is worth noting that cyanobacteria have evolved strategies for separating the essentially anaerobic reaction (such as biological nitrogen fixation) from the inevitable presence of molecular oxygen. Most commonly, the separation of the two incompatible reactions is accomplished by two manners: temporal separation following a diurnal cycle or spatial separation of specialized cells known as “heterocysts”.21,22 The natural anaerobic microenvironment formed by heterocysts may be recognized as a superior choice for efficient protection of heterologous oxygen-sensitive enzymes. Anabaena sp. strain PCC7120 (Anabaena PCC7120) is a typical nitrogen-fixing filamentous cyanobacte-

or the world’s limited fossil reserves and growing environmental concerns, it is becoming increasingly important to develop alternatives for sustainably producing chemicals and fuels from renewable resources.1,2 In the past decade, advancements in metabolic engineering and synthetic biology have increased our understanding of the diverse cellular systems that generate biofuels and chemicals from some abundant and inexpensive materials such as crude glycerol, lignin, or CO2.3,4 Compared to these heterotrophic hosts, cyanobacteria are more attractive hosts because of their ability to directly convert CO2 and solar energy into chemicals. In addition, availability of cyanobacterial genome sequences as well as various techniques and tools for gene manipulation have made cyanobacteria a useful host for bioengineering.5 Numerous efforts have been undertaken to develop engineered cyanobacteria to produce various value-added products including but not limited to ethanol,6 succinate,7 glycerol,8 1butanol,9 2,3-butanediol,10 squalene,11 isoprene,12 natural products,13 and limonene.14 Notably, most genes of introduced pathways are from heterotrophic bacteria and some enzymes of the pathways are even sensitive to oxygen, which require anaerobic conditions.9,15,16 Currently, one of the major limitation to photosynthetic production of desired chemicals is considered as the incompatibility between introduced heterologous © XXXX American Chemical Society

Received: June 7, 2018

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DOI: 10.1021/acssynbio.8b00239 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology

Figure 1. The simplified illustration of 1,3-PDO photosynthesis from CO2 with a genetically engineered filamentous nitrogenfixation cyanobacteriumAnabaena PCC7120. The left ellipse represents an oxygenic vegetative cell while the right one is an oxygen-free heterocyst. The color of blue presents oxygenic cellular environment of the vegetative cells and the color of light orange presents oxygen-free microenvironment of the heterocyst. The green lines show artificial biosynthetic pathway that was introduced into the engineered Anabaena PCC7120. Abbreviations: DAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; 3-HPA, 3-hydroxypropionaldehyde; 1,3-PDO, 1,3propanediol; GPP, glycerol-3-phosphatase; GDHt, glycerol dehydratase; YqhD, oxidoreductase; OPP pathway, oxidative pentose phosphate pathway; PSI, photosystem I; PSII, photosystem II.

27.0 mg L−1 when cultured in the BG11 medium. The results suggested that expression of oxygen-sensitive enzymes in the heterocysts is a potential alternative for improving the compatibility between the introduced pathways and oxygenic cyanobacteria.

rium that can differentiate the specialized cells “heterocysts” when grown diazotrophically in the absence of combined nitrogen.21 Furthermore, as the one of model cyanobacteria, many genetic engineering approaches for Anabaena PCC7120 have also been developed for genetic engineering.23−27 Therefore, it will be a practicable and potential strategy to tackle the incompatibility of oxygen-sensitive enzymes by insulating them in Anabaena PCC7120 heterocysts. 1,3-Propanediol (1,3-PDO) is an important chemical used for the synthesis of novel polyesters (such as polytrimethylene terephthalate) with superior stretching characteristics, cosmetics, lubricants, and drugs.28 Current strategies for 1,3-PDO production are costly and inefficient, and accumulate many other byproducts.29,30 Therefore, developing sustainable and environment-friendly technology for 1,3-PDO production has become increasingly important. In our previous study, autotrophic production of 1,3-PDO was first demonstrated by coculturing the glycerol-producing engineered Synechococcus elongatus PCC7942 with a native 1,3-PDO producer Klebsiella pneumoniae.8 Although 1,3-PDO production from CO2 using a single engineered cyanobacterial strain was achieved by introducing the enzymes for both glycerol synthesis from CO2 and glycerol reduction to 1,3-PDO.16 However, in this synthetic pathway, glycerol dehydration is catalyzed by the enzyme GDHt, which is oxygen-sensitive. The oxidative intracellular environment limits the efficiency of the oxygen-sensitive pathway in cyanobacteria. Therefore, it will be promising to use the proposed strategy based on compartmentalization (such as heterocysts) to encapsulate and protect the oxygen-sensitive enzyme. In this study, we constructed a heteroexpression pathway in Anabaena PCC7120 to synthesize 1,3-PDO directly from CO2 and solar energy (Figure 1). First, genes from different sources were tested to investigate their performance in S. elongatus PCC7942 and then the better cassette KP (containing the genes from K. pneumoniae) was introduced into Anabaena PCC7120. The engineered Anabaena PCC7120 strain P11 accumulated 46.0 mg L−1 of 1,3-PDO when heterocysts were present, which is approximately 1.7-fold higher as compared to

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RESULTS Genes from the Facultative Anaerobe Show Better Performance. The biological pathway of 1,3-PDO production from glycerol has been demonstrated in many microorganisms, such as Lactobacillus sp., Klebsiella sp., Citrobacter sp., and Clostridia sp.29,30 Among these, K. pneumoniae and Clostridium butyricum are two representatives of the good natural producers.29 Accordingly, we constructed the biosynthetic pathway of 1,3-PDO in cyanobacteria to synthesize 1,3-PDO directly from CO2 and solar energy (Figure 2a). Cassette KP consists of a glycerol-3-phosphatase (GPP, encoded by gpp) from Saccharomyces cerevisiae, a glycerol dehydratase (GDHtKP, encoded by dhaBCE) with a GDHt reactivating factor (GRFKP, encoded by dhaF, dhaG) from facultative anaerobe K. pneumoniae, and an oxidoreductase (YqhD, encoded by yqhD) from Escherichia coli, which was selected for its high activity toward 1,3-PDO and its ability to utilize both NADP+ and NAD+. In addition, GDHtKP and GRFKP were substituted with GDHtCB (encoded by dhaB1) and GRFCB (encoded by dhaB2) from a strictly anaerobic bacterium C. butyricum, forming a new assembled synthesis cassette referred to as CB, which is more oxygen-sensitive in cyanobacteria. As a model strain, S. elongatus PCC7942 was engineered to produce various chemicals from CO2 directly, including commodity products and high-value compounds.15,31 In this study, the cassettes for 1,3-PDO production were introduced into S. elongatus PCC7942 as a control trial. Strains P01 and P02 were constructed by recombination of plasmids pAM-BCE and pAM-B into S. elongatus PCC7942, respectively (Figure S1). The engineered strains were cultured stationarily in the BG11 medium with constant light exposure (100 μE s−1 m−2). The concentration of 1,3-PDO was detected by gas chromatography−mass spectrometry and reached a peak of B

DOI: 10.1021/acssynbio.8b00239 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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PCC7120 and transformants were confirmed by colony PCR (Figure S2a). In order to confirm the transcriptional levels of the heterologous genes of strain P11, quantitative reverse transcription PCR (RT-qPCR) was performed (Figure 4a). As shown in Figure 4a, gpp, yqhD, dhaF, dhaG, and dhaBCE were all successfully transcribed under the induction of IPTG. As for the different DNA transcriptional levels of the heterologous genes, this may have been caused by different arrangements and sources of the genes.8 The resulting engineered strain P11 was cultured stationarily in the BG11 medium with constant light exposure. 1,3-PDO production was sustained for 20 days, and the highest titer reached 27.0 mg L−1 on the 16th day (Figure 3b). No 1,3-PDO was detected in the culture of wildtype strain Anabaena PCC7120 (7120-WT). Furthermore, the engineered strain P11 was cultured stationarily in the BG10 medium to induce the formation of heterocysts with constant light exposure. 1,3-PDO production was sustained for 20 days, and the highest titer reached 46.0 mg L−1, which is approximately 1.7-fold higher than that cultured in the BG11 medium (Figure 3b). These results indicated that when the oxygen-sensitive enzyme of 1,3-PDO synthesis was encapsulated in the heterocysts, the specialized microaerobic environment helped to protect the oxygen-sensitive enzyme from oxygen and improved its’ activity under oxygenic photosynthesis. Additionally, 1,3-PDO accumulation suggested that a part of carbon flux of strain P11 was redirected to the target product synthesis. Although P11 cultures showed a decrease in cell density compared to the wild-type strain (Figure 3c), the production of 1,3-PDO biomass in strain P11 resulted in increased biomass production at varying extents during this process. Moreover, the concentration of the intermediate product glycerol was lower when heterocysts were present than that of no heterocysts (Figure S3). This revealed that strain P11 was superior for glycerol conversion when heterocysts were present. Hence, increasing supply of the intermediate product, glycerol, and cell growth may further improve 1,3PDO yield. Optimization of 1,3-PDO Production in Engineered Anabaena PCC7120. As for strain P11, comparing with that of no heterocysts, the final product 1,3-PDO was higher and the intermediate product glycerol was lower when heterocysts were present. Additionally, strain P11 showed a decrease in cell density compared to the wild-type strain. Therefore, the concentration of the intermediate product and the lower cell density may have limited the accumulation of 1,3-PDO. Besides, Anabaena PCC7120 can grow in the presence of exogenous glucose, which can improve the cell growth evidently of Anabaena PCC7120.32 To further enhance the 1,3-PDO titer, we evaluated the effect of glycerol and glucose addition on the synthesis of 1,3-PDO and cell density. The concentration of added glycerol was 5 g L−1 and glucose was 3 g L−1, unless otherwise specified. Strain P11 was cultured stationarily with constant light exposure with glycerol addition. Increased 1,3-PDO accumulation was achieved when strain P11 was cultured in the BG10 medium, showing a high concentration of 306 mg L−1, while when strain P11 was cultured in the BG11 medium resulted in a value of 251 mg L−1 (Figure 5a), which is approximately 1.2-fold higher than that cultured in the BG11 medium. This tend is analogous to what we observed without intermediate product addition (Figure 3b). These results indicate that the bottleneck changed to the relative lower concentration of intermediate product

Figure 2. 1,3-PDO production by the engineered S. elongatus PCC7942 strains P01 and P02. (a) Schematic representation of inserting genes integration into S. elongatus PCC7942 genome at Neutral Site I (NSI). gpp, coding gene of glycerol-3-phosphatase from S. cerevisiae; dhaB1, coding gene of glycerol dehydratase and dhaB2, coding gene of glycerol dehydratase reactivating factor from C. butyricum; yqhD, coding gene of 1,3-PDO oxidoreductase isoenzyme from E. coli; dhaBCE, coding gene of glycerol dehydratase and dhaF, dhaG, coding gene of glycerol dehydratase reactivating factor from K. pneumoniae. (b) 1,3-PDO accumulation of engineered strains P01 and P02 incubated stationarily with constant light exposure. Asterisk marks indicate the levels of statistical significance (* means p-value ≤ 0.05, and ** means p-value ≤ 0. 001). Error bars indicate SD (n = 3).

26.9 mg L−1 and 2.0 mg L−1 in cultures of P01 and P02 on the 16th day, respectively. In contrast, 1,3-PDO was not detected in the culture of wild-type strain S. elongatus PCC7942 (7942WT) (Figure 2b). Thus, the two artificial cassettes introduced into S. elongatus PCC7942 were functional. However, the cassette KP containing genes from the facultative anaerobe K. pneumoniae was much more efficient than the cassette CB which contains genes from strictly anaerobic bacterium C. butyricum. It has been suggested that cyanobacteria have an oxygenic cellular environment, the production and efficiency of introduced heterologous pathways in cyanobacteria are limited because of the enzyme oxygen-sensitivity.9,18,19 Here, the performance of the two 1,3-PDO production cassettes in S. elongatus PCC7942 further indicated that intracellular dissolved oxygen may have an important influence on oxygen-sensitive pathway in cyanobacteria. Thus, approaches for improving the efficiency of oxygen-sensitive heterologous pathways in cyanobacteria are urgently needed. Spatial Segregation Enhances 1,3-PDO Production by Heterocysts. Anabaena PCC7120 is a filamentous, nitrogen-fixing cyanobacterium, and received increasing attention recently. To achieve photosynthetic production of 1,3-PDO in Anabaena PCC7120, we constructed the plasmid pRL277Y-BCE, which contains genes of cassette KP tandemly arrayed under the control of the isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter Ptrc (Figure 3a). Plasmid pRL277Y-BCE was then introduced into Anabaena C

DOI: 10.1021/acssynbio.8b00239 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Figure 3. 1,3-PDO production by the engineered Anabaena PCC7120 strain P11. (a) Schematic representation of gpp, dhaBCE, dhaFG, yqhD and Nm genes integration into noncoding region FR of Anabaena PCC7120. gpp, coding gene of glycerol-3-phosphatase from S. cerevisiae; dhaBCE, coding gene of glycerol dehydratase and dhaF, dhaG, coding gene of glycerol dehydratase reactivating factor from K. pneumoniae; yqhD, coding gene of 1,3-PDO oxidoreductase isoenzyme from E. coli; Nm, encoding Neomycin resistance marker. (b) 1,3-PDO accumulation of engineered strain P11 incubated stationarily with constant light exposure. (c) Total biomass production per day of Anabaena PCC7120 WT (open circle) and P11 (closed square) strains incubated stationarily with constant light exposure. Cell biomass was determined as cell dry weight. Total biomass was cell biomass plus 1,3-PDO produced. Asterisk marks indicate the levels of statistical significance (* means p-value ≤ 0.05, and ** means p-value ≤ 0.001). Error bars indicate SD (n = 3).

Figure 4. Quantitative reverse-transcription PCR analysis of relative transcriptional levels of foreign genes in strains P11 (a) and P12 (b). All data were normalized by the housekeeping gene rnpB. Asterisk marks indicate the levels of statistical significance (n.s. means p-value > 0.05 and * means p-value ≤ 0.05). Error bars indicate SD (n = 3).

Figure 5. Optimization of 1,3-PDO production in engineered Anabaena PCC7120. (a) 1,3-PDO accumulation of engineered strain P11 incubated in stationary culture, with glycerol (gly, 5 g L−1) addition. (b) 1,3-PDO accumulation of concentrated strain P11 incubated in stationary culture, with glycerol (gly, 5 g L−1) and glucose (glu, 3 g L−1) addition. Asterisk marks indicate the levels of statistical significance (n.s. means p-value > 0.05 and * means p-value ≤ 0.05). Error bars indicate SD (n = 3).

There is no clear difference in the final 1,3-PDO titer when strain P11 was cultured in BG11 (257 mg L−1), while there was a striking increase in 1,3-PDO accumulation (594 mg L−1) when strain P11 was cultured in the BG10 medium. To determine whether glucose was directly used for 1,3-PDO production, 13C6-glucose (all six carbon atoms in the glucose

when the heterocysts relieved the oxygen toxicity to GDHt in the engineered strain. To improve the cell density, we added glucose as an exogenous carbon substrate and glycerol to ensure sufficient intermediate product in the medium for P11 (Figure 5b). As expected, the cell growth of strain P11 was improved evidently. D

DOI: 10.1021/acssynbio.8b00239 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology were 13C labeled from Sigma) was used to verify if the carbon skeleton of 1,3-PDO was originated from the added glucose. The mass spectrum of 1,3-PDO produced from the 13C6glucose medium matches well with that from the glucose medium (Figure S4). This suggest that glucose addition does not contribute much to 1,3-PDO photosynthetic production. Furthermore, the number of heterocysts of strain P11 with glucose addition or not was determined by counts of more than 3000 cells by staining and then checked by light microscope. After glucose addition the frequency of heterocysts was increased approximately from 8.01% to 8.62% and more contiguous heterocysts were observed (Figure S5). These results indicate that the direct cause of the increase in 1,3-PDO was not glucose addition, but rather the anaerobic protection for GDHt and the efficient conversion of glycerol in heterocysts. The Extremely Oxygen-Sensitive Enzyme Is Active in Heterocysts. C. butyricum is an excellent native 1,3-PDO producer from glycerol and the only reported microorganism containing a coenzyme B12-independent glycerol dehydratase.33 The deficiency of coenzyme B12 is one of the key limitations to improving 1,3-PDO titer. However, the efficiency of cassette CB is lower than that of cassette KP in S. elongatus PCC7942. This indicates that the genes form obligate anaerobes may be much more sensitive to the oxidative intracellular environment of cyanobacteria. It is necessary to test the performance of cassette CB in Anabaena PCC7120, particularly when heterocysts exist. Thus, we constructed plasmid pRL277Y-B which contains genes of cassette CB tandemly arrayed under the control of the IPTG inducible promoter Ptrc (Figure 6a). Next, pRL277Y-B was introduced into Anabaena PCC7120 and transformants were confirmed by colony PCR (Figure S2b). The resulting engineering strain P12 was cultured stationarily in the BG11 and the BG10 media with constant light exposure, respectively. 1,3-PDO production was sustained for 20 days, but no 1,3-PDO was detected when P12 was cultured in the BG11. We detected the 1,3-PDO accumulation (approximately 1.59 mg L−1) on day 16 when P12 was cultured in the BG10 (Figure 6b). To optimize 1,3PDO production and avoid the using of IPTG, we replaced the inducible promoter Ptrc with the strong constitutive promoter PpsbA in Anabaena PCC7120 to create plasmid pRL277Y-P.34 Next, pRL277Y-P was introduced into Anabaena PCC7120 and transformants were confirmed by colony PCR (Figure S2c). The resulting engineered strain P13 was cultured stationarily in the BG11 and BG10 media with constant light exposure, respectively. The same results were observed that 1,3-PDO was detected only when heterocysts were present (Figure 6b). To supply sufficient intermediate product, strain P12 was cultured with glycerol and cell growth conditions were improved by glucose addition (Figure 6b). Surprisingly, as shown in Figure 6b, varying extents of 1,3-PDO accumulation were detected only when heterocysts were present. As for cassette CB, the key step was conversion of glycerol to 1,3-PDO, which was catalyzed by GDHtCB, an oxygen-sensitive enzyme from a strictly anaerobic strain. 1,3-PDO synthesis of the engineered strains P12 and P13 were detected only when heterocysts were present. In order to investigate whether all introduced heterologous genes of strain P12 were expressed when P12 was cultured in the BG11, quantitative reverse transcription PCR (RT-qPCR) was performed (Figure 4b). As shown in Figure 4b, gpp, yqhD, dhaB1, and dhaB2 were successfully transcribed under the induction of IPTG and the

Figure 6. 1,3-PDO production by the engineered Anabaena PCC7120 strain P12, P13, and P14. (a) Schematic representation of gpp, dhaB1, dhaB2, yqhD and Nm genes integration into noncoding region FR of Anabaena PCC7120. gpp, coding gene of glycerol-3-phosphatase from S. cerevisiae; dhaB1, coding gene of glycerol dehydratase and dhaB2, coding gene of glycerol dehydratase reactivating factor from C. butyricum; yqhD, coding gene of 1,3-PDO oxidoreductase isoenzyme from E. coli; Nm, encoding Neomycin resistance marker. (b) Cell density and 1,3-PDO titer of engineered strains incubated in stationary culture under different conditions. Medium (11) indicates that the strain was cultured in the BG11 medium, while the medium (10) indicates that the strain was cultured in the BG10 medium. Glycerol/Glucose (+) indicates that 5 g L−1 glycerol/3 g L−1 glucose was added to the medium, while () indicates glycerol/glucose was not added to the medium. Error bars indicate SD (n = 3).

transcriptional level of the same gene was comparable when strain P12 was cultured in the BG11 and BG10 media, respectively. This revealed that there might be some other reasons that could result in no 1,3-PDO production of the strain P12 when it was cultured in the BG11. Therefore, it is logical to suggest that the protection of natural oxygen-free compartment formed by the heterocyst was important for the key oxygen-sensitive of GDHtCB here. For further confirming the protective effect of heterocysts on cassette CB, we constructed plasmid pRL277Y-N in which the promoter Ptrc was substituted with promoter Pnif, which is active only in the heterocysts.35 Plasmid transformation or strain construction was performed as described above. The engineered strain P14 was then performed under standard oxygenic incubation with constant light exposure. We detected the 1,3-PDO accumulation of strain P14 (0.33 mg L−1) only when cells were cultured in the BG10 medium (Figure 6b). These results confirmed that due to the incompatibility of oxygen-sensitivity, enzymes originating from anaerobes are often difficult to be active in cyanobacteria. Hence, it is reasonable to speculate that GDHt CB was inactivated easily in the oxygenic cyanobacteria cells and the natural oxygen-free compartment formed by the heterocyst was necessary for it. E

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Figure 7. Effects of glycerol (a) and 1,3-PDO (b) on cell growth of Anabaena PCC7120 wild type strain. Various concentrations of chemicals were added into the culture medium when the cell density of the cultures reached OD730nm of 0.4−0.7, and the growth was monitored for the next few days. Error bars indicate SD (n = 3).

Tolerance of Anabaena PCC7120 to the Intermediate and End-Product. Accumulation of products may restrain the growth of cells and prevent higher production of target chemical. Hence, we assessed the effects of adding glycerol and 1,3-PDO to Anabaena PCC7120 on cell growth (Figure 7). For the tolerance experiments, glycerol and 1,3-PDO were added to the medium at different concentrations, respectively. Slight growth inhibition was observed in the presence of 20 g L−1 glycerol and 10 g L−1 1,3-PDO. However, the highest titers of products obtained in the present study did not approach lethal levels. Thus, the effects of all chemicals were negligible on Anabaena PCC7120 in this study and 1,3-PDO production in cyanobacteria may be further improved.

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inherent dissolved oxygen with an anaerobic pathway in cyanobacteria. Moreover, the frequency of heterocysts of Anabaena PCC7120 can be increased significantly or up to nearly 100% by disrupting or overexpression differentiationrelated control genes.39,40 Thus, the further improvements in 1,3-PDO synthesis in Anabaena PCC7120 could be expected by increasing the concentration of intermediate product or the frequency of heterocysts. Additionally, as the differentiated cells of Anabaena PCC7120 heterocysts are induced by nitrogen-deficient condition, both inducer and nitrogen source are not required in the production process of using the engineered Anabaena PCC7120. As well-known, suppling nitrogen imposes a predominant cost in industrial scale biological production. Therefore, using engineered Anabaena PCC7120 for photosynthetic production is also attractive and promising in terms of cost-efficiency, and sustainability. Many previous studies confirmed that because of enzyme oxygen sensitivity, pathways originating from anaerobes often do not function efficiently in oxygenic cyanobacteria.17 However, searching for an alternative enzyme that is oxygentolerant or reforming enzymes by protein engineering is laborintensive. Herein, the natural compartmentalization by cell differentiation provide a wise substitution of enzyme mining, minimizing cost and time spent on enzyme screening or engineering. Spatial segregation by dividing the synthetic pathway into different microorganisms and then combining the whole pathway by coculture of engineered organisms to overcome the incompatibility of the introduced pathway and the chassis have been investigated.8,41 However, constructing synthetic coculture system requires the complex designing to control the intercellular interactions, spatiotemporal coordination, robustness, stability and biocontainment of synthetic microbial communities.42 Since heterocysts are the naturally differentiated cells, the ratio of heterocysts and vegetative cells is well controlled at a constant level. Intercellular channels are also existed between heterocysts and vegetative cells for the effective exchange of molecules including metabolites and intercellular signals.21 Hence, spatial segregation by using the heterocysts is considered much convenient and delicate. As shown in Figure 6, when the cassette CB containing coenzyme B12-independent GDHtCB and GRFCB from strictly anaerobic bacterium C. butyricum was introduced into Anabaena PCC7120, 1,3-PDO was detected only when heterocysts were present. While the transcriptional level of the introduced genes were comparable when strain P12 was cultured in the BG11 and BG10 media, respectively. These results suggested that the toxicity of dissolved oxygen in the cytochylema could even completely inactivate the extremely

DISCUSSION

In the past decade, with the developments and applications of metabolic engineering and synthetic biology, various chemicals have been produced by microorganisms which equipped with a naturally or artificially assembled synthetic metabolic pathway.4,36 This approach has generated significant improvements in chemical production, but the incompatibility between the introduced pathway and the chassis host is often encountered as a major challenge of metabolic engineering and synthetic biology.37 In nature, organisms tend to accomplish various incompatible reactions or intricate functions by spatial organization which is important for preventing the escape of volatile or toxic intermediates and creating private cofactor pools.38 Hence, it may be helpful to sequester specific metabolic pathways by spatial compartmentalization to overcome these incompatibilities in synthetic biology, particularly those requiring a specialized microenvironment. In this work, we constructed engineered cyanobacterial strains for 1,3-PDO synthesis and realized spatial segregation by heterocysts, aiming to overcome the incompatibility of the key oxygen-sensitive enzyme (GDHt) and the dissolved oxygen. As expected, the engineered Anabaena PCC7120 strain P11 showed approximately 1.7-fold higher titers when heterocysts were present than that of no heterocysts. Besides, a striking increase in 1,3-PDO accumulation (594 mg L−1) was achieved only when heterocysts were present after glucose addition (Figure 5b). After glucose addition, the frequency of heterocysts was increased and more contiguous heterocysts were observed. Concurrently, analysis of 1,3-PDO produced from the glucose medium by isotope labeling experiment indicated that glucose addition does not contribute much to 1,3-PDO production here. These results further indicate that the spatial compartmentalization formed by heterocysts is an effective strategy to overcome the incompatibility of the F

DOI: 10.1021/acssynbio.8b00239 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology Table 1. Bacterial Strains Used in This Study strain

characteristic(s)a

E. coli DH5α E. coli H10B(pRL623+pRL443) E. coli BL21(DE3) Saccharomyces cerevisiae K. pneumoniae ATCC 25955 C. butyricum DSM 10702 Anabaena sp. strain PCC7120 S. elongatus PCC7942 P01 P02 P11 P12 P13 P14

Cloning host Contains plasmid pRL623 and pRL443, Cmr, Tcr, and Specr Source of yqhD gene Source of gpp gene Source of genes dhaBCE, dhaF, and dhaG Source of genes dhaB1 and dhaB2 Wild type Wild type Ptrc::gpp, Ptrc::dhaBCE, Ptrc::dhaFG, Ptrc::yqhD, integrated into NSI, Specr Ptrc::gpp, Ptrc::dhaB1, Ptrc::dhaB2, Ptrc::yqhD, integrated into NSI, Specr Ptrc::gpp, Ptrc::dhaBCE, Ptrc::dhaFG, Ptrc::yqhD, Nm, integrated into FR, Nmr Ptrc::gpp, Ptrc::dhaB1, Ptrc::dhaB2, Ptrc::yqhD, Nm, integrated into FR, Nmr PpsbA::gpp, PpsbA::dhaB1, PpsbA::dhaB2, PpsbA::yqhD, Nm, integrated into FR, Nmr Pnif::gpp, Pnif::dhaB1, Pnif::dhaB2, Pnif::yqhD, Nm, integrated into FR, Nmr

a

reference or source Novagen 49

Novagen Lab collection ATCC DSM 39

ATCC 33912 This study This study This study This study This study This study

Nmr, Tcr, Specr, and Cmr: resistance to neomycin, tetracycline, spectinomycin and chloramphenicol, respectively.

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CONCLUSIONS This study describes a strategy for solving the problem of incompatibility in synthetic biology using the natural compartmentalization formed by heterocysts. The cassettes of oxygen-sensitive 1,3-PDO biosynthetic pathway were packaged into heterocysts of Anabaena PCC7120 to protect them from oxygen. As expected, when heterocysts presented, the 1,3-PDO accumulation by the engineered Anabaena PCC7120 strain P11 was approximately 1.7-fold higher than that without the presence of heterocysts. Moreover, as for the strains (P12, P13, and P14) containing cassette CB, encoding extremely oxygen-sensitive 1,3-PDO synthesis pathway, the product 1,3-PDO could only be detected when heterocysts were present. These results demonstrate that the specialized anaerobic microenvironment formed by heterocysts is effective for keeping the activity of the key oxygen-sensitive enzyme GDHt. This study highlights the potential of heterocysts that may serve as a general spatial compartmentalization to overcome the incompatibility between anaerobic pathway(s) and cyanobacteria. This work is a successful exploration of spatial segregation by taking advantage of naturally evolved compartmentalization strategy. The proposed strategy may serve as an alternative promising way for bypassing the incompatibility that is increasingly impedimental in the design and construction of artificial biological systems.

oxygen-sensitive enzyme GDHtCB and the anaerobic microenvironment formed by the heterocysts is necessary to keep the activity of GDHtCB. This strategy would be critically useful for extremely oxygen-sensitive enzymes introduced in cyanobacterial engineering. In the photoproduction processes of n-butanol the oxidative intracellular environment of cyanobacteria significantly hindered the efficiency of the introduced pathways.19,20 They optimized the process by using an exhaustive strategy in which different trial and error steps was included, such as light condition, oxygen condition of culturing system, and inhibitor of photosystem. Screening the oxygen tolerant enzyme from different sources to replace the oxygen-sensitive one was also performed. In this study, a new strategy of using natural compartmentalization was successfully achieved for compatibly expressing the oxygen-sensitive pathway in oxidative cyanobacteria, without time-consuming construction and tedious coculture engineering. This strategy has the potential of serving as a universal strategy that could be especially useful for the anaerobic pathway introduced. Moreover, the final step of our introduced pathway is NADPH-driving. When cultured in the BG10 medium, Anabaena PCC7120 induced the formation of heterocysts. In heterocysts, carbon reductants from vegetative cells were metabolized to generate carbon skeletons, large amounts of NADPH for nitrogen fixation, and maintaining a microaerobic environment.43 Thus, it is reasonable to suggest that the abundant reducing power also contributed to the improvement of 1,3-PDO synthesis. This is another advantage of using heterocysts as the natural compartmentalization. It is worth noting that in nature the spatial organization is also achieved by membrane bound organelles of eukaryotes, protein “organelles” (bacterial microcompartments) of prokaryotes and multienzyme complexes besides specialized cells (heterocysts).44,45 These manners of spatial organization could also be used for compartmentalization in metabolic engineering and synthetic biology,46,47 but much deeper exploitations at many scales are still required. Although technological and cognitive challenges remain to be overcome, the spatial segregation that mimic natural strategies hold great potential in metabolic engineering and synthetic biology. This study will increase the understanding of native cellular strategies of spatial organization and provide approaches for further design of biosynthetic systems.

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METHODS Chemicals, Strains and Culture Conditions. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise specified. The FastPfu DNA polymerase and pEASY-Uni Seamless Cloning and Assembly Kit were acquired from TransGen Biotech (Beijing, China). The restriction enzymes were purchased from New England Biolabs (Beverly, MA, USA). Oligonucleotide synthesis was carried out by Generay Biotech (China). Genomic DNA was extracted using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). Strains used in this study are shown in Table 1. S. elongatus PCC7942, Anabaena PCC7120 and their derivatives were grown in the BG11 medium (1.5 g L−1 NaNO3, 0.075 g L−1 MgSO4·7H2O, 0.039 g L−1 K2HPO4, 0.027 g L−1 CaCl2, 0.020 g L−1 Na2CO3, 0.006 g L−1 citrate, 0.006 g L−1 ferric ammonium citrate, 0.001 g L−1 Na2EDTA·2H2O, 2.860 mg G

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ACS Synthetic Biology L−1 H3BO3, 1.810 mg L−1 MnCl2·4H2O, 0.390 mg L−1 Na2MoO4·2H2O, 0.220 mg L−1 ZnSO4·7H2O, 0.080 mg L−1 CuSO4·5H2O, and 0.050 mg L−1 Co(NO3)2·6H2O). To induce heterocysts formation, Anabaena PCC7120 and its derivatives were grown in the BG10 medium (the BG11 medium without combined nitrogen NaNO3, N2-fixing condition). Heterocysts are stained with 0.5% Alcian blue (w/v) for 5 min and then checked by light microscope (Figure S6). Antibiotic was added into the medium as required, unless otherwise specified. The working concentration of antibiotics were as follows: spectinomycin (Spec) 2 μg mL−1 and streptomycin (Sm) 2 μg mL−1 or neomycin (Nm) 20 μg mL−1. For stationary culture, Anabaena PCC7120 was grown at 30 °C, using the 100 mL BG11 or BG10 medium in a 250 mL Erlenmeyer flask at 30 °C under continuous illumination with an illumination intensity of 100 μE s−1 m−2. Growth of Anabaena PCC7120 was monitored by measuring the optical density at 730 nm. Cell biomass was monitored by measuring the dry cell weight of Anabaena PCC7120. Escherichia coli strains were grown in Luria−Bertani (LB) medium with appropriate antibiotics when required. Plasmids were constructed in E. coli DH5α strains for propagation and storage. E. coli H10B (contains plasmids pRL623, pRL443 and pRL277Y or its derivatives) was used for conjugation with Anabaena PCC7120. The working concentration of antibiotics were as follows: Spec 100 μg mL−1, chloromycetin (Cm) 20 μg mL−1, tetracycline (Tc) 25 μg mL−1 or Nm 100 μg mL−1. Plasmid Construction. Plasmids used and constructed in this study are listed in Table S1. All connection between gene and plasmid fragments were achieved by recombinationligation technique as described, unless otherwise specified. All genes and promoter sequence were confirmed by sequencing. Primers used for plasmid construction in this study are listed in Table S2. The plasmid pRL277 (GenBank NO: L05082.1), which cannot replicate in Anabaena PCC7120, was chosen for construction of the basic integrative expression vector pRL277Y comprised of flanking regions F and R for integration (Figure S7). The 946 bp F region and the 1071 bp R region (for flanking) were selected from an intergenic, noncoding region of Anabaena PCC7120 genome.24 The F and R fragment were PCR amplified from Anabaena PCC7120 DNA using the primer pairs F-F/F-R (SpeI) and R-F (SpeI)/RR, respectively. Then the two fragments were ligated to pRL277 vector at SpeI sites via the recombination-ligation technique by pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, China), producing a 8.823 kb integrative expression vector pRL277Y. Plasmid pAM-BCE was constructed by the insertion of gpp, dhaBCE, dhaFG, yahD under the control of the IPTGinducible promoter Ptrc into pAM-MCS12. The genes gpp, yqhD, dhaBCE, dhaFG were amplified by PCR with the genomic DNA of S. cerevisiae, K. pneumoniae and E. coli BL21 (DE3) as templates, respectively. The gpp gene was amplified with the primers gpp-F/gpp-R and its related promoter Ptrc was amplified with the primers Ptrc-gpp-F/Ptrc-gpp-R by high-fidelity PCR and then were cloned into the XhoI sites of pAM-MCS12 to construct pAM01. The yqhD gene was amplified with the primers yqhD-F/yqhD-R and its related promoter Ptrc was amplified with the primers Ptrc-yqhD-F/Ptrc-yqhD-R and then gene yqhD and promoter Ptrc were cloned into the BglII sites of pAM01 to construct pAM02. The gene dhaBCE was amplified with primers dhaBCE-F/dhaBCE-R and cloned into the EcoRI

sites of pAM02 to construct pAM03. The gene dhaF, dhaG and promoter Ptrc fragments were amplified with primers dhaF-F/ dhaF-R, dhaG-F/dhaG-R and Ptrc-dhaF-F/Ptrc-dhaF-R, respectively and then were cloned into the BamHI site of pAM03 to construct pAM-BCE. Plasmid pAM-B was constructed by the insertion of dhaB1 and dhaB2 under the control of the IPTGinducible promoter Ptrc into pAM02. The genes dhaB1, dhaB2 were amplified by high-fidelity PCR with the genomic DNA of C. butyricum. The gene dhaB1 was amplified from with primers dhaB1-F/dhaB1-R and cloned into the EcoRI sites of pAM02 to construct pAM04. Finally, the gene dhaB2 was amplified with primers dhaB2-F/dhaB2-R and promoter Ptrc fragments was amplified with primers Ptrc-dhaB2-F/Ptrc-dhaB2-R and then were cloned into the BamHI site of pAM04 to construct pAMB. Plasmid pRL277Y-BCE was constructed by the insertion of gpp, dhaBCE, dhaFG, yahD under the control of the IPTGinducible promoter Ptrc and Nm into the speI site of pRL277Y. The genes gpp, yqhD, dhaBCE, dhaFG and Nm were amplified by high-fidelity PCR with plasmid pAM-BCE and pET-28a (+) as templates, using four pairs of primers P (Ptrc)-F/ptrcBCE (BamHI), Ptrc (yqhD)-F/yqhD (Ptrc)-R, Nm (yqhD)-F/Nm (PM)-R and Ptrc (PM)-F/dhaFG (Ptrc)-R, respectively. Plasmid pRL277Y-B was constructed by the insertion of gpp, dhaB1, dhaB2, yahD under the control of the IPTG-inducible promoter Ptrc and Nm into the speI site of pRL277Y, using three primers P (Ptrc)-F/yqhD (Ptrc)-R, Nm (yqhD)-F/Nm (R)R and gpp (Ptrc)-F/dhaB2 (F)-R, respectively. The genes dhaB1, dhaB2, promoter Pnif and PpsbA were amplified by PCR with plasmid pAM-B, the genomic DNA of Anabaena PCC7120 and plasmid pRL439, respectively. Plasmid pRL277Y-PpsbAB was constructed by the insertion of gpp, dhaB1, dhaB2, yahD under the control of the constitutive promoter PpsbA and Nm into pRL277Y. The gpp gene was amplified with the primers gpp (PpbsA)-F/gpp (PpbsA)-R and its related promoter PpsbA was amplified with the primers PpbsA (gpp)-F/PpbsA (gpp)-R by high-fidelity PCR. Then gene gpp and promoter PpsbA were cloned into the EcoRI sites of pUC-19 to construct p101. The yqhD gene was amplified with the primers yqhD (PpbsA)-F/yqhD (PpbsA)-R, its related promoter PpsbA was amplified with the primers PpbsA (yqhD)-F/PpbsA (yqhD)-R and Nm was amplified with the primers Nm-F/ Nm-R by high-fidelity PCR. Then promoter PpsbA, gene yqhD and Nm were cloned into the BamHI sites of p101 to construct p102. The dhaB1 gene was amplified with the primers B1 (PpsbA)-F/B1 (PpsbA)-R and its related promoter PpsbA was amplified with the primers PpsbA (B1)-F/PpsbA (B1)-R by highfidelity PCR. Then gene dhaB1 and promoter PpsbA were cloned into the EcoRI sites of pUC-19 to construct p103. The dhaB2 gene was amplified with the primers dhaB2 (PpsbA)-F/ dhaB2 (PpsbA)-R and its related promoter PpsbA was amplified with the primers PpsbA (dhaB1)-F/PpsbA (dhaB1)-R by highfidelity PCR. Then promoter PpsbA and gene yqhD were cloned into the BamHI site of p103 to construct p104.The gene fragments of p102 and p104 were amplified with the primers P1-F/P1(Nm)-R and P2(Nm)-F/P2-R respectively, and then were cloned into speI site of pRL277Y to construct plasmid pRL277Y-PpsbAB. Plasmid pRL277Y-Pnif B was constructed using the same procedure described above with plasmid pRL277Y-PpsbAB by replacing promoter PpsbA with promoter Pnif, which is active only in the heterocysts. H

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ACS Synthetic Biology Construction of Engineered S. elongatus PCC7942. The strains used and constructed are listed in Table 1. Plasmids pAM-BCE and pAM-B were integrated into the S. elongatus PCC7942 genome by homologous recombination and got strains P01 and P02, respectively. S. elongatus PCC7942 was transformed using protocols previously described.48 All transformants were selected on BG11 agar plates supplemented with spectinomycin (Spec) 20 μg mL−1. All inserted genes integrated into the chromosome of S. elongatus PCC7942 were confirmed by colony PCR (Figure S1). Construction of Engineered Anabaena PCC7120. The strains used and constructed are listed in Table 1. All plasmids which contain genes for 1,3-PDO photosynthetic production were transferred into the wild-type Anabaena PCC7120 strain by conjugal transfer, using biparental mating system of Anabaena PCC7120 and E. coli H10B26,49 with the following modifications as described below. The inserted gene cassette was integrated into the Anabaena PCC7120 genome by homologous recombination at noncoding region (FR region). E. coli H10B bearing helper (pRL623) and conjugal (pRL443) plasmids was transformed by the integrate plasmid (pRL277Y) and selected on LB plates containing appropriate antibiotics for the selection of three plasmids. Selected colonies were grown overnight in 5 mL of LB containing appropriate antibiotics, subcultured by adding 30 μL of overnight culture to 30 mL of fresh LB containing appropriate antibiotics. Cells were harvested (OD620nm of 0.6−0.8), by centrifugation at 6000g for 2 min, washed three times with 10 mL of LB to completely remove antibiotics. The harvested cells were resuspended with 0.5 mL LB and then mixed with Anabaena PCC7120. A 30 mL culture of wild-type (WT) Anabaena PCC7120 was grown to exponential stage (OD730 nm of 0.4−0.7). Cells were harvested by centrifugation at 5000g for 10 min, washed three times with 10 mL fresh BG11 medium. The harvested cells were resuspended in 0.5 mL of fresh BG11, and then mixed with the above E. coli H10B harboring three plasmids for conjugal transfer. This mixture was placed stationarily under lighted conditions at 30 °C for 4 h. The 100 μL conjugal mixture was transferred on to an autoclaved nitrocellulose filter placed on a BG11 with 10% LB agar plate and grown stationarily with continuous illumination for 2 days at 30 °C. The filter was then transferred to a BG11 plate containing Spec and Sm (2 μg mL−1, respectively) to select for positive exconjugants. Individual exconjugants were further purified three times by sonicated for 60−180 s to break filaments into 1−4 cell lengths which were confirmed under light microscopy. The breaking filaments were restreaked onto fresh BG11 plates containing 25 μg mL−1 Nm for the selection of successful recombinants. The correct recombinants were confirmed by colony PCR to verify integration of target genes into the chromosome (Figure S2). RT-qPCR. Anabaena PCC7120 strains were grown in 100 mL of the BG11 or BG10 medium in a 250 mL shake flask under light condition at 30 °C under continuous illumination with an illumination intensity of 100 μE s−1 m−2. When the cell density of the Anabaena PCC7120 cultures reached to 0.4−0.7 (OD730 nm), 1.0 mM IPTG was added to induce the expression of the target genes. Total RNA was extracted using the RNAprep Pure Cell/Bacteria kit (Tiangen Biotech, China). Total RNA was treated with Thermo Scientific DNase I and quantified using Nanodrop 2000, then used as a template for

cDNA synthesis with random primers and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The rnpB housekeeping gene was used as a control. Primers for quantitative PCR were designed using Beacon Designer software (Biosoft International, Palo Alto, California, USA) (Table S3). Quantitative RT-PCR was performed using the SuperReal PreMix SYBR Green kit (Tiangen Biotech, China) and the CFX96 Real-Time system (Bio-Rad, Hercules, CA, USA). Analytical Techniques. The concentrations of glycerol and 1,3-PDO were determined by gas chromatography−mass spectrometry (GC−MS, Agilent 6850/5975C system) or highperformance liquid chromatography (HPLC, Agilent 1200 series). The GC−MS system was equipped with a capillary column (HP-5 ms; 30 m × 0.25 mm) via the autosampler. The GC−MS analysis of 1,3-PDO and glycerol in culture broth samples was derived via silylation using protocols previously described (Figure S8).50 GC−MS data were processed using ChemStation software (Agilent). The HPLC system was equipped with a Bio-Rad Aminex HPX-87H column (300 × 7.8 mm) and refractive index and UV/vis detectors was used for detection of the concentrations of glycerol and 1,3-PDO. The analysis was performed at 55 °C with a mobile phase of 5 mM H2SO4 at a flow rate of 0.5 mL min−1. Culture samples (2 mL) were centrifuged for 5 min at 12 000g. The supernatant was used for product analysis. Data Process and Analysis. All the experiments were performed at least three biological replicates for assay. A oneway ANOVA method was used for the statistical analysis of different experiments settings, such as the effect of heterocysts and the glycerol/glucose addition on 1,3-PDO production. Figures were drawn by origin 8 and Adobe Illustrator CC 2015. The level of statistical significance is represented by asterisk marks in the according figures.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00239. Information for plasmids and strains construction as well as chemical analysis; Figure S1−S8; Table S1−S3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-34206647. Fax: +86-21-34206723. E-mail: [email protected]. ORCID

Ping Xu: 0000-0002-4418-9680 Author Contributions

H.L., F.T. and P.X. conceived the study. H.L. performed the experiments. H.L. and J.N. analyzed the data. H.L. wrote the manuscript. F.T. and P.X. revised the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The plasmids pRL277, pRL623, pRL443, and pRL439 were kindly provided by Professor C. P. Wolk and Xudong Xu. Anabaena sp. strain PCC7120 was kindly provided by I

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(20) Lan, E. I., and Liao, J. C. (2011) Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide. Metab. Eng. 13, 353−363. (21) Kumar, K., Mella-Herrera, R. A., and Golden, J. W. (2010) Cyanobacterial heterocysts. Cold Spring Harbor Perspect. Biol. 2, a000315. (22) Berman-Frank, I., Lundgren, P., and Falkowski, P. (2001) Segregation of Nitrogen Fixation and Oxygenic Photosynthesis in the Marine Cyanobacterium Trichodesmium. Science 294, 1534−1537. (23) Higo, A., Isu, A., Fukaya, Y., and Hisabori, T. (2017) Designing Synthetic Flexible Gene Regulation Networks Using RNA Devices in Cyanobacteria. ACS Synth. Biol. 6, 55−61. (24) Chaurasia, A. K., Parasnis, A., and Apte, S. K. (2008) An integrative expression vector for strain improvement and environmental applications of the nitrogen fixing cyanobacterium, Anabaena sp. strain PCC7120. J. Microbiol. Methods 73, 133−141. (25) Elhai, J., and Wolk, C. P. (1988) A versatile class of positiveselection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68, 119−138. (26) Halfmann, C., Gu, L., Gibbons, W., and Zhou, R. (2014) Genetically engineering cyanobacteria to convert CO2, water, and light into the long-chain hydrocarbon farnesene. Appl. Microbiol. Biotechnol. 98, 9869−9877. (27) Cai, Y., and Wolk, C. P. (1990) Use of a Conditionally Lethal Gene in Anabaena sp. Strain PCC 7120 To Select for Double Recombinants and To Entrap Insertion Sequences. J. Bacteriol. 172, 3138−3145. (28) Xin, B., Tao, F., Wang, Y., Liu, H., Ma, C., and Xu, P. (2017) Coordination of metabolic pathways: Enhanced carbon conservation in 1,3-propanediol production by coupling with optically pure lactate biosynthesis. Metab. Eng. 41, 102−114. (29) Saxena, R. K., Anand, P., Saran, S., and Isar, J. (2009) Microbial production of 1,3-propanediol: Recent developments and emerging opportunities. Biotechnol. Adv. 27, 895−913. (30) Celinska, E. (2010) Debottlenecking the 1,3-propanediol pathway by metabolic engineering. Biotechnol. Adv. 28, 519−530. (31) Oliver, N. J., Rabinovitch-Deere, C. A., Carroll, A. L., Nozzi, N. E., Case, A. E., and Atsumi, S. (2016) Cyanobacterial metabolic engineering for biofuel and chemical production. Curr. Opin. Chem. Biol. 35, 43−50. (32) Guoce, Y., Dingji, S., Zhaoling, C., Wei, C., and Fan, O. (2011) Growth and Physiological Features of Cyanobacterium Anabaena sp. Strain PCC 7120 in a Glucose-Mixotrophic Culture. Chin. J. Chem. Eng. 19, 108−115. (33) Gonzalez-Pajuelo, M., Meynial-Salles, I., Mendes, F., Andrade, J. C., Vasconcelos, I., and Soucaille, P. (2005) Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1,3propanediol from glycerol. Metab. Eng. 7, 329−336. (34) Elhai, J. (1993) Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120. FEMS Microbiol. Lett. 114, 179−184. (35) Bauer, C. C., and Haselkorn, R. (1995) Vectors for Determining the Differential Expression of Genes in Heterocysts and Vegetative Cells of Anabaena sp. Strain PCC 7120. J. Bacteriol. 177, 3332−3336. (36) Lee, J. W., Na, D., Park, J. M., Lee, J., Choi, S., and Lee, S. Y. (2012) Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat. Chem. Biol. 8, 536−546. (37) Lechner, A., Brunk, E., and Keasling, J. D. (2016) The Need for Integrated Approaches in Metabolic Engineering. Cold Spring Harbor Perspect. Biol. 8, a023903. (38) Frank, S., Lawrence, A. D., Prentice, M. B., and Warren, M. J. (2013) Bacterial microcompartments moving into a synthetic biological world. J. Biotechnol. 163, 273−279. (39) Yoon, H.-S., and Golden, J. W. (1988) Heterocyst Pattern Formation Controlled by a Diffusible Peptide. Science 282, 935−938. (40) Khudyakov, I. Y., and Golden, J. W. (2004) Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena

Professor J. W. Golden. This work was supported by the grant from Science and Technology Commission of Shanghai Municipality (17JC1404800), and the grant from the National Natural Science Foundation of China (31570101).

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REFERENCES

(1) Singh, V., Chaudhary, D. K., Mani, I., and Dhar, P. K. (2016) Recent advances and challenges of the use of cyanobacteria towards the production of biofuels. Renewable Sustainable Energy Rev. 60, 1− 10. (2) Ni, J., Gao, Y. Y., Tao, F., Liu, H. Y., and Xu, P. (2018) Temperature-Directed Biocatalysis for the Sustainable Production of Aromatic Aldehydes or Alcohols. Angew. Chem., Int. Ed. 57, 1214− 1217. (3) Sun, X., Shen, X., Jain, R., Lin, Y., Wang, J., Sun, J., Wang, J., Yan, Y., and Yuan, Q. (2015) Synthesis of chemicals by metabolic engineering of microbes. Chem. Soc. Rev. 44, 3760−3785. (4) Rabinovitch-Deere, C. A., Oliver, J. W., Rodriguez, G. M., and Atsumi, S. (2013) Synthetic biology and metabolic engineering approaches to produce biofuels. Chem. Rev. 113, 4611−4632. (5) Hagemann, M., and Hess, W. R. (2017) Systems and synthetic biology for the biotechnological application of cyanobacteria. Curr. Opin. Biotechnol. 49, 94−99. (6) Gao, Z., Zhao, H., Li, Z., Tan, X., and Lu, X. (2012) Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria. Energy Environ. Sci. 5, 9857− 9865. (7) Lan, E. I., and Wei, C. T. (2016) Metabolic engineering of cyanobacteria for the photosynthetic production of succinate. Metab. Eng. 38, 483−493. (8) Wang, Y., Tao, F., Ni, J., Li, C., and Xu, P. (2015) Production of C3 platform chemicals from CO2 by genetically engineered cyanobacteria. Green Chem. 17, 3100−3110. (9) Lan, E. I., and Liao, J. C. (2012) ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proc. Natl. Acad. Sci. U. S. A. 109, 6018−6023. (10) Nozzi, N. E., Case, A. E., Carroll, A. L., and Atsumi, S. (2017) Systematic Approaches to Efficiently Produce 2,3-Butanediol in a Marine Cyanobacterium. ACS Synth. Biol. 6, 2136−2144. (11) Choi, S. Y., Wang, J. Y., Kwak, H. S., Lee, S. M., Um, Y., Kim, Y., Sim, S. J., Choi, J. I., and Woo, H. M. (2017) Improvement of Squalene Production from CO2 in Synechococcus elongatus PCC 7942 by Metabolic Engineering and Scalable Production in a Photobioreactor. ACS Synth. Biol. 6, 1289−1295. (12) Chaves, J. E., Rueda-Romero, P., Kirst, H., and Melis, A. (2017) Engineering Isoprene Synthase Expression and Activity in Cyanobacteria. ACS Synth. Biol. 6, 2281−2292. (13) Ni, J., Tao, F., Wang, Y., Yao, F., and Xu, P. (2016) A photoautotrophic platform for the sustainable production of valuable plant natural products from CO2. Green Chem. 18, 3537−3548. (14) Halfmann, C., Gu, L., and Zhou, R. (2014) Engineering cyanobacteria for the production of a cyclic hydrocarbon fuel from CO2 and H2O. Green Chem. 16, 3175−3185. (15) Angermayr, S. A., Gorchs Rovira, A., and Hellingwerf, K. J. (2015) Metabolic engineering of cyanobacteria for the synthesis of commodity products. Trends Biotechnol. 33, 352−361. (16) Hirokawa, Y., Maki, Y., Tatsuke, T., and Hanai, T. (2016) Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway. Metab. Eng. 34, 97−103. (17) Gudmundsson, S., and Nogales, J. (2015) Cyanobacteria as photosynthetic biocatalysts: a systems biology perspective. Mol. BioSyst. 11, 60−70. (18) Latifi, A., Ruiz, M., and Zhang, C. C. (2009) Oxidative stress in cyanobacteria. FEMS Microbiol. Rev. 33, 258−278. (19) Lan, E. I., Ro, S. Y., and Liao, J. C. (2013) Oxygen-tolerant coenzyme A-acylating aldehyde dehydrogenase facilitates efficient photosynthetic n-butanol biosynthesis in cyanobacteria. Energy Environ. Sci. 6, 2672−2681. J

DOI: 10.1021/acssynbio.8b00239 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology sp. PCC 7120, can be separated by mutation. Proc. Natl. Acad. Sci. U. S. A. 101, 16040−16045. (41) Jones, J. A., and Wang, X. (2018) Use of bacterial co-cultures for the efficient production of chemicals. Curr. Opin. Biotechnol. 53, 33−38. (42) Johns, N. I., Blazejewski, T., Gomes, A. L., and Wang, H. H. (2016) Principles for designing synthetic microbial communities. Curr. Opin. Microbiol. 31, 146−153. (43) Bö hme, H. (1998) Regulation of nitrogen fixation in heterocyst-forming cyanobacteria. Trends Plant Sci. 3, 346−351. (44) Agapakis, C. M., Boyle, P. M., and Silver, P. A. (2012) Natural strategies for the spatial optimization of metabolism in synthetic biology. Nat. Chem. Biol. 8, 527−535. (45) Mampel, J., Buescher, J. M., Meurer, G., and Eck, J. (2013) Coping with complexity in metabolic engineering. Trends Biotechnol. 31, 52−60. (46) Slininger Lee, M. F., Jakobson, C. M., and Tullman-Ercek, D. (2017) Evidence for Improved Encapsulated Pathway Behavior in a Bacterial Microcompartment through Shell Protein Engineering. ACS Synth. Biol. 6, 1880−1891. (47) Chen, A. H., and Silver, P. A. (2012) Designing biological compartmentalization. Trends Cell Biol. 22, 662−670. (48) Golden, S. S., Brusslan, J., and Haselkorn, R (1987) Genetic engineering of the cyanobacterial chromosome. Methods Enzymol. 153, 215−231. (49) Elhai, J., Verpritskiy, A., Muro-Pastor, A. M., Flores, E., and Wolk, C. P. (1997) Reduction of Conjugal Transfer Efficiency by Three Restriction Activities of Anabaena sp. Strain PCC 7120. J. Bacteriol. 179, 1998−2005. (50) Dai, L., Tao, F., Tang, H. Z., Guo, Y. L., Shen, Y. L., and Xu, P. (2017) Directing enzyme devolution for biosynthesis of alkanols and 1,n-alkanediols from natural polyhydroxy compounds. Metab. Eng. 44, 70−80.

K

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