Qingdao Energy has made a series of progress in photosynthetic biosynthesis of ethanol from cyanobacteria

Ethanol is the largest renewable bio-liquid fuel with the highest degree of application. At this stage, the main source of bioethanol is the use of bio-refinery processes that use sugar-rich agricultural biomass as raw materials. The “maize ethanol” is the most representative, but it’s “grabbing with food and competing with others”. The raw material supply model has caused great social controversy; Cellulosic ethanol synthesis technology using lignocellulosic and other agricultural and forestry wastes as raw materials has alleviated the shortage of “grain ethanol” in the supply of raw materials, but the pretreatment of cellulose raw materials and The enzymatic saccharification process consumes a lot of energy, water and cellulase, which greatly increases the production cost. Compared with the biorefinery process, the CO2 To Ethanol (CTE) technology that directly converts CO2 and solar energy into ethanol through the photosynthetic microbiological platform reduces raw material pretreatment, the loss of the substrate extraction process, and also saves fresh water and land The demand shows greater potential and advantages in terms of economy and sustainability.

The microbial metabolism engineering team of the Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences has made a series of research progresses in the cyanobacterium photosynthetic biosynthetic ethanol technology. The team used the important model cyanobacteria Synechocystis sp. PCC6803 as a chassis strain, and the PdcZM-AdhIIZM (pyruvate decarboxylase-type II alcohol dehydrogenase) pathway from Zymomonas mobilis into PCC6803. , Opened the ethanol photosynthesis route, and achieved the synthesis and secretion of ethanol in the engineering strains. Based on the analysis of the physiological and metabolic background characteristics of the cyanobacteria, the NADPH-dependent type II alcohol dehydrogenase gene slr1192 from Synechocystis sp. PCC6803 itself was used. After replacing AdhIIZM, the adaptability of the ethanol synthesis pathway to the algae strains was successfully optimized, and the ethanol yield was increased by 50%. On the basis of this strategy, a combination of competitive pathway knockout and metabolic pathway copy number enhancement strategies was finally obtained. The syn-HZ24, a strong algae photosynthetic synthesizing capacity, was cultivated in a column reactor for 28 days. The ethanol yield reached 5.5 g/L, and the synthesis rate was 0.2 g/L/day, which was the leading international level. The above results laid the basis for the team's research in the direction of cyanobacterial ethanol and has been published in Energy & Environmental Science (Gao, et al, 2012, 5:9857–9865).

In order to further enhance the potential of ethanol photosynthetic synthesis in cyanobacterial-engineered algae strains, the team used in vitro remodeling and dynamic analysis strategies to dig deeper into the catalytic properties of the newly developed PdcZM-slr1192 pathway. First, the pathway was rebuilt from the complex intracellular environment extracellularly to purify the enzyme (PdcZM/slr1192), substrate (pyruvate, Pyruvate), cofactor (NADPH/NADH/TPP), metal ion (Mg2+) The titration analysis of the catalytic efficiency of the entire pathway and the intermediate product (acetaldehyde) showed that the limiting factor affecting ethanol synthesis was PdcZM instead of slr1192; under the condition of constant total protein concentration, PdcZM-slr1192 When the concentration ratio is 4:6, the whole route has the largest catalytic activity for ethanol synthesis. In order to verify the results obtained under Cell free conditions, the researchers constructed an engineering strain with different PdcZM-slr1192 concentrations in Synechocystis sp. PCC6803. Through metabolic engineering, combined with enzyme activity and protein content analysis experiments, the existing engineering strains were confirmed. The expression level and activity of PdcZM are the primary limiting factors for ethanol synthesis ability. In addition, the titration data in in vitro reconstitution system combined with the actual content analysis of intracellular metabolites of cyanobacteria also showed that increasing the supply of NADPH and pyruvic acid should also be an important choice for increasing the efficiency of ethanol synthesis. The above results clarify the direction of further transformation of cyanobacterial ethanol photosynthetic engineering algae strains and have been published in Bioenergy for Biofuels (Luan, et al, 2015, 8:184).

Based on the cyanobacterial ethanol photosynthetic engineering algae strains that have been developed, the team has further explored the expansion culture technology of the photosynthetic cell factory. Photosynthetic organisms are often manufactured outdoors, under open, non-sterile conditions, and are therefore often exposed to serious threats from various forms of biological contamination, which in turn leads to failure to expand cultivation. Researchers found that the synthesis and accumulation process of ethanol was seriously affected by microbial contamination in the open and large-scale cultivation of the engineered algae strain Syn-HZ24. Through analysis and identification, Pannonibacter phragmitetus was identified as the main threat source of ethanol photosynthetic synthesis. The bacteria can grow with ethanol as the sole carbon source, rapidly consume the ethanol synthesized by the engineering algae strain and multiply in the culture system. Through the physiological and biochemical analysis of Pannonibacter phragmitetus and Syn-HZ24, the researchers proposed to increase the pH value of the culture system to inhibit the invasion of Pannonibacter phragmitetus and restore the photosynthetic synthesis of ethanol (Bicarbonate-based Integrated Carbon Capture System, BICCS). The results were validated under both the column reactor and the outdoor film bag system. The results show that this strategy can effectively solve the biological pollution problem of Syn-HZ24 in the open culture process. After 9 days of large-scale cultivation, the ethanol yield reaches 0.9 g. /L, but there was no ethanol accumulation in the control system under conventional pH conditions. This part of the work identified a new model of bio-pollution in the process of large-scale cultivation of engineered cyanobacteria, and specifically designed and validated the solution, which has universal reference significance for the engineering and large-scale development of plant cultivation of photosynthetic cells. This work was recently published in Biotechnology for Biofuels (Zhu, et al, 2017, 10:93).

The above research was supported by the National Natural Science Foundation of China, the "863" Plan, the Taishan Scholars Program of Shandong Province, the Natural Science Foundation of Shandong Province, and the Qingdao Talent Innovation Project.