Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae.

Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142g/L with production yield of 0.89g/g and productivity of 3.55gL(-1)h(-1) under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast.

Engineering cellular redox balance in Saccharomyces cerevisiae for improved production of L-lactic acid.

Owing to the growing market for the biodegradable and renewable polymer, polylactic acid, world demand for lactic acid is rapidly increasing. However, the very high concentrations desired for industrial production of the free lactic acid create toxicity and low pH concerns for manufacturers. Saccharomyces cerevisiae is the most well characterized eukaryote, a preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust, commercially compatible workhorse to be exploited for the production of diverse chemicals. S. cerevisiae has also been explored as a host for lactic acid production because of its high acid tolerance. Here, we constructed an L-lactic acid-overproducing S. cerevisiae by redirecting cellular metabolic fluxes to the production of L-lactic acid. To this end, we deleted the S. cerevisiae genes encoding pyruvate decarboxylase 1 (PDC1), L-lactate cytochrome-c oxidoreductase (CYB2), and glycerol-3-phosphate dehydrogenase (GPD1), replacing them with a heterologous L-lactate dehydrogenase (LDH) gene. Two new target genes encoding isoenzymes of the external NADH dehydrogenase (NDE1 and NDE2), were also deleted from the genome to re-engineer the intracellular redox balance. The resulting strain was found to produce L-lactic acid more efficiently (32.6% increase in final L-lactic acid titer). When tested in a bioreactor in fed-batch mode, this engineered strain produced 117 g/L of L-lactic acid under low pH conditions. This result demonstrates that the redox balance engineering should be coupled with the metabolic engineering in the construction of L-lactic acid-overproducing S. cerevisiae.

Fed-batch culture of astaxanthin-rich Haematococcus pluvialis by exponential nutrient feeding and stepwise light supplementation.

A fed-batch culture process followed by subsequent photoautotrophic induction was established for the high density culture of astaxanthin-rich Haematococcus pluvialis using a CO(2)-fed flat type photobioreactor under unsynchronized illumination. Fed-batch culture was performed with an exponential feeding strategy of the growth-limiting nutrients, nitrate and phosphate, concurrently with the stepwise supplementation of light depending on the cell concentration. During the growth phase, a biomass of 1.47 g/L was obtained at a biomass productivity of 0.33 g/L/day. Photoautotrophic induction of the well-grown vegetative cells was performed consecutively by increasing the light intensity to 400 micromol photon/m(2)/s, while keeping the other conditions in the CO(2)-fed flat type photobioreactor fixed, yielding an astaxanthin production of 190 mg/L at an astaxanthin productivity of 14 mg/L/day. The proposed sequential photoautotrophic process has high potential as simple and productive process for the production of valuable Haematococcus astaxanthin.

Non-labeled detection of waterborne pathogen Cryptosporidium parvum using a polydiacetylene-based fluorescence chip.

A non-labeling fluorescence sensor system was developed using polydiacetylene (PDA) liposomes composed of 10,12-pentacosadiynoic acid (PCDA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at a 8:2 molar ratio. The PDA liposomes were immobilized onto an amine-coated glass surface using peptide bonding between the carboxyl group of the liposome and the amine group of the glass surface. The optimum ratio of the cross linker (NHS/EDC) to PDA liposome was determined to be 50% for strong immobilization of the liposomes. Residual carboxyl groups of the PDA liposomes were selectively biotinylated, followed by sequential binding of streptavidin and biotin-antibody (bioreceptor). Finally, the performance of the PDA liposome chip was tested for detecting Cryptosporidium parvum, and yielded a detection limit of 1 x 10(3) oocysts/mL. From these results, it is expected that the PDA liposome chip will have high application potential for the detection of waterborne pathogens including C. parvum.

Surface plasmon resonance-based inhibition assay for real-time detection of Cryptosporidium parvum oocyst.

A surface plasmon resonance (SPR)-based inhibition assay method using a polyclonal anti-mouse IgM arrayed Cryptosporidium sensor chip was developed for the real-time detection of Cryptosporidium parvum oocysts. The Cryptosporidium sensor chip was fabricated by subsequent immobilization of streptavidin and polyclonal anti-mouse IgM (secondary antibody) onto heterogeneous self-assembled monolayers (SAMs). The assay consisted of the immunoreaction step between monoclonal anti-C. parvum oocyst (primary antibody) and oocysts, followed by the binding step of the unbound primary antibody onto the secondary antibody surface. It enhanced not only the immunoreaction yield of the oocysts by batch reaction but also the accessibility of analytes to the chip surface by antibody-antibody interaction. Furthermore, the use of optimum concentration of the primary antibody maximized its binding response on the chip. An inversely linear calibration curve for the oocyst concentration versus SPR signal was obtained in the range of 1x10(6)-1x10(2)oocystsml(-1). The oocyst detection was also successfully achieved in natural water systems. These results indicate that the SPR-based inhibition assay using the Cryptosporidium sensor chip has high application potential for the real-time analysis of C. parvum oocyst in laboratory and field water monitoring.

Direct extraction of astaxanthin from Haematococcus culture using vegetable oils.

A green, downstream process using common vegetable oils was used for the direct extraction of astaxanthin from Haematococcus. The process consists of a single integrated unit to extract astaxanthin with subsequent separation of the astaxanthin-containing oil extract. Without a cell harvest process step, the culture broth was directly mixed with the vegetable oils; the astaxanthin inside the cell was extracted into the vegetable oil phase by hydrophobic interactions, with recovery yields of 88% and above. The oil extracts were simply separated from the culture medium containing cell debris by gravity settling only.

Selective extraction of free astaxanthin from Haematococcus culture using a tandem organic solvent system.

A novel tandem solvent process of dodecane and methanol was developed for the selective extraction of free astaxanthin from red encysted Haematococcus culture. The process consists of dodecane extraction for astaxanthin mixture from the culture (stage 1) and methanol extraction for free astaxanthin from the dodecane extract (stage 2). In the first stage, astaxanthin mixture was directly extracted to dodecane from the culture broth without cell harvest process, followed by a rapid separation of the dodecane extract and the culture medium containing cell debris by simple settling. In the second stage, free astaxanthin was selectively collected to methanol from the dodecane extract, accompanied with saponification of astaxanthin-esters by the addition of NaOH to methanol. During saponification, use of the optimum NaOH concentration (0.02 M) and low temperature (4 degrees C) reaction minimized the degradation of free astaxanthin, resulting in a total recovery yield of free astaxanthin of over 85%. The free-astaxanthin-containing methanol extract was also simply separated from dodecane by gravity settling, after which the astaxanthin-free dodecane was effectively recycled to the first stage, yielding a stable extractability of astaxanthin mixture during repeated extraction. Our results indicate the potential of the proposed tandem solvent process as an alternative extraction technology for the high-value antioxidant Haematococcus astaxanthin.