Co-production of acetoin and succinic acid by metabolically engineered Enterobacter cloacae.

BACKGROUND: Renewable chemicals have attracted attention due to increasing interest in environmental concerns and resource utilization. Biobased production of industrial compounds from nonfood biomass has become increasingly important as a sustainable replacement for traditional petroleum-based production processes depending on fossil resources. Therefore, we engineered an Enterobacter cloacae budC and ldhA double-deletion strain (namely, EC∆budC∆ldhA) to redirect carbon fluxes and optimized the culture conditions to co-produce succinic acid and acetoin. RESULTS: In this work, E. cloacae was metabolically engineered to enhance its combined succinic acid and acetoin production during fermentation. Strain EC∆budC∆ldhA was constructed by deleting 2,3-butanediol dehydrogenase (budC), which is involved in 2,3-butanediol production, and lactate dehydrogenase (ldhA), which is involved in lactic acid production, from the E. cloacae genome. After redirecting and fine-tuning the E. cloacae metabolic flux, succinic acid and acetoin production was enhanced, and the combined production titers of acetoin and succinic acid from glucose were 17.75 and 2.75 g L-1, respectively. Moreover, to further improve acetoin and succinic acid production, glucose and NaHCO3 modes and times of feeding were optimized during fermentation of the EC∆budC∆ldhA strain. The maximum titers of acetoin and succinic acid were 39.5 and 20.3 g L-1 at 72 h, respectively. CONCLUSIONS: The engineered strain EC∆budC∆ldhA is useful for the co-production of acetoin and succinic acid and for reducing microbial fermentation costs by combining processes into a single step.

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.

Characterization of a heat-tolerant Chlorella sp. GD mutant with enhanced photosynthetic CO2 fixation efficiency and its implication as lactic acid fermentation feedstock.

BACKGROUND: Fermentative production of lactic acid from algae-based carbohydrates devoid of lignin has attracted great attention for its potential as a suitable alternative substrate compared to lignocellulosic biomass. RESULTS: A Chlorella sp. GD mutant with enhanced thermo-tolerance was obtained by mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine to overcome outdoor high-temperature inhibition and it was used as a feedstock for fermentative lactic acid production. The indoor experiments showed that biomass, reducing sugar content, photosynthetic O2 evolution rate, photosystem II activity (Fv/Fm and Fv'/Fm'), and chlorophyll content increased as temperature, light intensity, and CO2 concentration increased. The mutant showed similar DIC affinity and initial slope of photosynthetic light response curve (α) as that of the wild type but had higher dissolved inorganic carbon (DIC) utilization capacity and maximum photosynthesis rate (Pmax). Moreover, the PSII activity (Fv'/Fm') in the mutant remained normal without acclimation process after being transferred to photobioreactor. This suggests that efficient utilization of incident high light and enhanced carbon fixation with its subsequent flux to carbohydrates accumulation in the mutant contributes to higher sugar and biomass productivity under enriched CO2 condition. The mutant was cultured outdoors in a photobioreactor with 6% CO2 aeration in hot summer season in southern Taiwan. The harvested biomass was subjected to separate hydrolysis and fermentation (SHF) for lactic acid production with carbohydrate concentration equivalent to 20 g/L glucose using the lactic acid-producing bacterium Lactobacillus plantarum 23. The conversion rate and yield of lactic acid were 80% and 0.43 g/g Chlorella biomass, respectively. CONCLUSIONS: These results demonstrated that the thermo-tolerant Chlorella mutant with high photosynthetic efficiency and biomass productivity under hot outdoor condition is an efficient fermentative feedstock for large-scale lactic acid production.

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.

Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus.

Microalgal strains of Scenedesmus obliquus have the great potential for the production of biofuels, CO2 fixation, and bioremediation. However, metabolic engineering of S. obliquus to improve their useful phenotypes are still not fully developed. In this study, S. obliquus strain CPC2 was genetically engineered to promote the autotrophic growth and lipid productivity. The overexpression plasmid containing the type 2 diacylglycerol acyltransferse (DGAT) gene DGTT1 from Chlamydomonas reinhardtii was constructed and transformed into S. obliquus CPC2, and the positive transformants were obtained. The expression of DGTT1 gene was confirmed by reverse transcription PCR analysis. Enhanced lipid content of the transformant S. obliquus CPC2-G1 by nearly two-fold was observed. The biomass concentration of the recombinant strains was also 29% higher than that of the wild-type strain. Furthermore, the recombinant strain CPC2-G1 was successfully grown in 40 L tubular type photobioreactor and open pond system in an outdoor environment. The lipid content, biomass concentration, and biomass productivity obtained from 40 L tubular PBR were 127.8% 20.0%, and 232.6% higher than those obtained from the wild-type strain. The major aim of this work is to develop a tool to genetically engineer an isolated S. obliquus strain for the desired purpose. This is the first report that genetic engineering of S. obliquus has been successful employed to improve both the microalgal cell growth and the lipid production.

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.