Engineering acetylation platform for the total biosynthesis of D-amino acids.

Optically pure D-amino acids are key chemicals with various applications. Although the production of specific D-amino acids has been achieved by chemical synthesis or with in vitro enzyme catalysts, it is challenging to convert a simple carbon source into D-amino acids with high efficiency. Here, we design an artificial metabolic pathway by engineering bacteria to heterologously express racemase and N-acetyltransferase to produce N-acetyl-D-amino acids from L-amino acids. This new platform allows the cytotoxicity of D-amino acids to be avoided. The universal potential of this acetylation protection strategy for effectively synthesizing optically pure D-amino acids is demonstrated by testing sixteen amino acid targets. Furthermore, we combine pathway optimization and metabolic engineering in Escherichia coli and achieve practically useful efficiency with four specific examples, including N-acetyl-D-valine, N-acetyl-D-serine, N-acetyl-D-phenylalanine and N-acetyl-D-phenylglycine, with titers reaching 5.65 g/L, 5.25 g/L, 8.025 g/L and 130 mg/L, respectively. This work opens up opportunities for synthesizing D-amino acids directly from simple carbon sources, avoiding costly and unsustainable conventional approaches.

Isolation and characterization of Chlorella sp. mutants with enhanced thermo- and CO2 tolerances for CO2 sequestration and utilization of flue gases.

BACKGROUND: The increasing emission of flue gas from industrial plants contributes to environmental pollution, global warming, and climate change. Microalgae have been considered excellent biological materials for flue gas removal, particularly CO2 mitigation. However, tolerance to high temperatures is also critical for outdoor microalgal mass cultivation. Therefore, flue gas- and thermo-tolerant mutants of Chlorella vulgaris ESP-31 were generated and characterized for their ability to grow under various conditions. RESULTS: In this study, we obtained two CO2- and thermo-tolerant mutants of Chlorella vulgaris ESP-31, namely, 283 and 359, with enhanced CO2 tolerance and thermo-tolerance by using N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagenesis followed by screening at high temperature and under high CO2 conditions with the w-zipper pouch selection method. The two mutants exhibited higher photosynthetic activity and biomass productivity than that of the ESP-31 wild type. More importantly, the mutants were able to grow at high temperature (40 °C) and a high concentration of simulated flue gas (25% CO2, 80-90 ppm SO2, 90-100 ppm NO) and showed higher carbohydrate and lipid contents than did the ESP-31 wild type. CONCLUSIONS: The two thermo- and flue gas-tolerant mutants of Chlorella vulgaris ESP-31 were useful for CO2 mitigation from flue gas under heated conditions and for the production of carbohydrates and biodiesel directly using CO2 from flue gas.

Improvement of outdoor culture efficiency of cyanobacteria by over-expression of stress tolerance genes and its implication as bio-refinery feedstock.

This study was undertaken to increase the biomass and carbohydrate productivities of a freshwater cyanobacterium Synechococcus elongatus under hot outdoor conditions through genetic manipulation to facilitate the application of using the cyanobacterial biomass as bio-refinery feedstocks. The stress tolerance genes (hspA, osmotin) were expressed in S. elongatus to improve their growth under various environment stresses of outdoor cultivation. The results revealed that over-expression of hspA and osmotin significantly improved temperature (45°C), high light intensity, and salt tolerances of S. elongatus cells, making it capable of efficiently growing in seawater under outdoor cultivation. The carbohydrate productivity of these stress tolerant strains was also 15-30-fold higher than that of the control strain, although the carbohydrate contents of the recombinant and control strains were similar. Our findings demonstrate that the genetic engineering for improved stresses tolerance in S. elongatus could facilitate the feasibility of using cyanobacteria as feedstock for bio-refinery industry.

Using recombinant cyanobacterium (Synechococcus elongatus) with increased carbohydrate productivity as feedstock for bioethanol production via separate hydrolysis and fermentation process.

In this work, a recombinant cyanobacterium strain with increased photosynthesis rate, cell growth and carbohydrate production efficiency was genetically engineered by co-expressing ictB, ecaA, and acsAB (encoded for bacterial cellulose) in Synechococcus elongatus PCC7942. The resulting cyanobacterial biomass could be effectively hydrolyzed with dilute acid (2% sulfuric acid), achieving a nearly 90% glucose recovery at a biomass concentration of 80 g/L. Bioethanol can be produced from fermenting the acidic hydrolysate of S. elongatus PCC7942 via separate hydrolysis and fermentation (SHF) process at a concentration of 7.2 g/L and with a 91% theoretical yield.