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 slr0301gene. 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 CO2, 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 CO2 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 CO2 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% CO2-air (vol/vol) into the photo-bioreactor, the ethanol yield of 5.5 g L-1 was achieved with a high cell density (OD730≈15) after 26 days of fermentation [7]. In comparison, without using photo-bioreactor and pumping CO2, the strain SYN009 produced only 1.3 g L-1 of ethanol when the cell density was much lower, which OD730 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 OD730 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.