Co-factor engineering to increase ethanol production of Synechocystis sp.

Abstract

Environmental issues that have been warned for decades are now actually affecting our daily life in the form of climate change, emergence of new diseases, and the destruction of ecosystems. Excessive fossil fuel consumption is an undeniably major cause of these warning signals. Accordingly, alternative energy sources based on biomass appear increasingly attractive, and various candidates are appealing to replace the current energy supply system with little or no modification and without further pollution. Cyanobacteria are gaining great attention as promising renewable energy sources, contributing to production of clean bioenergy and CO2 mitigation at the same time. Their rapid growth rate, low land requirement for cultivation, natural diversity, and potential to genetic engineering offer great advantages over competing resources such as wood and agricultural crops/residues which are referred as 1st and 2nd generation of biomass. Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmospheric carbon dioxide by photosynthesis. Genetic manipulation has increased the variety of chemicals that cyanobacteria can produce. However, their uniquely abundant NADPH-pool, in other words insufficient supply of NADH, tends to limit their production yields in case of utilizing NADH-dependent enzyme, which is quite common in heterotrophic microbes. To overcome this co-factor imbalance, various co-factor engineering approaches have been employed, including reducing NADH-dependence of pathways by replacement with NADPH-utilizing enzymes or modification of co-factor specificity of enzymes. In this study, two co-factor engineering approaches were applied to increase ethanol production of the model unicellular cyanobacterium, Synechocystis sp. PCC 6803. Firstly, instead of NADH- dependent alcohol dehydrogenase, NADPH-dependent enzyme, YqhD from Escherichia coli was introduced with pyruvate decarboxylase (PDC) from Zymomonas mobilis. Secondly, the co-factor specificity of alcohol dehydrogenase from Zymomas mobilis was changed by introducing single and multiple mutations. In addition to those approaches, metabolic engineering to increase NADPH availability was attempted by activation of oxidative pentose phosphate pathway. It was expected that excessive NADPH would be a driving force to further increase the ethanol production via NADPH-utilizing pathways. By over-expression of zwf, G6PDH activity was increased about 4-fold and 3.4-fold compared to wild type under autotrophic and mixotrophic conditions respectively, and consequently, NADPH production was also increased. Increased NADPH production promoted biomass production. NADPH, as a major electron donor in cyanobacteria, is used for carbon fixation, ATP generation, respiration and various biosynthetic reactions. Thus, increased NADPH production by zwf over-expression probably aided those cellular activities including calvin cycle and respiratory electron transport chain, resulting in higher biomass production. The utilization of NADPH-dependent alcohol dehydrogenase, Yqhd, resulted in higher ethanol production compared to previous report using NADH-dependent enzyme in Synechocystis sp. PCC6803. This result clearly showed that NADPH-utilizing pathway is more favored than NADH-utilizing one in cyanobacteria. Moreover, zwf over-expression further increased ethanol production, and this result supported that utilization of NADPH-dependent enzyme and increase of the NADPH supply could creat a synergistic effect to improve ethanol production of cyanobacteria. Changing the co-factor specificity of alcohol dehydrogenase from Zymomonas mobilis were conducted by rational enzyme engineering approaches. The key residues determining the co-factor specificity were identified by comparative sequence analysis, and they were replaced by other residues through site-directed mutagenesis. Ethanol productions were increased in the single and triple mutation strains. The wild type enzyme rarely uses NADPH as its co-factor. Relaxing co-factor specificity by single mutation (D39G) enabled the enzyme to utilize both NADH and NADPH, leading to increase in ethanol production. Complete switch of co-factor specificity by triple mutations (D39G/F41S/M42S) further increased ethanol production, revealing that reducing NADH-dependence of enzyme could be beneficial in cyanobacteria. To increase NADPH supply, zwf over-expression was also attempted, however, the ethanol production was decreased possibly due to inhibition by NADPH. With the recent development of genetic manipulation tools and extensive research efforts, cyanobacteria have gained a great attention as a promising renewable energy producer. Genetically engineered cyanobacteria strains could successfully convert sunlight and carbon dioxide into valuable biofuels and chemicals. However, due to low productivity, there is still a long way to go for commercialization of cyanobacterial biofuel production. Introduction of heterologous genes is not the end, but its impacts on the intracellular environment should be considered together for the desirable result. Co-factor engineering to overcome the co-factor imbalance and increase the co-factor supply in cyanobacteria can be a smart strategy to create a synergy effect and improve cyanobacterial biofuel production efficiently. It is expected that the approaches used in this study can be applied to other cyanobacterial biofuel production, and enhance the production efficiency.