Biosynthesis of aliphatic plastic monomers with amino residues in Yarrowia lipolytica.

Introduciton: The α,ω-diamines (NH2-(CH2)n-NH2) and ω -amino fatty acids (NH2-(CH2)n-COOH) have been widely used as building blocks in polymerindustries. Medium- to long-chain (C8 to C18) fatty acid monomers with amino residues are almost exclusively produced via chemical processes that generate hazardous waste and induce severe environmental problems, such as global warming and pollution. Here, we present the construction platformstrains of Yarrowia lipolytica a cheese-ripening yeast, for direct biotransformation of hydrocarbons into medium- to long-chain α,ω-diamines and ωamino fatty acids using metabolic engineering of endogenous fatty acid ω- and ß-oxidation pathways and introducing heterologous ω-transaminase in Y. lipolytica. Methods: We deleted six genes encoding the acyl-CoA oxidase (ACO1-6) and four fatty aldehyde dehydrogenase genes (FALDH1-4), which catalyze fatty acid ß-oxidation and downstream oxidation of fatty aldehydes in Y. lipolytica, respectively. The ω-transaminase from Chromobacterium violaceum DSM30191 was introduced into the genome of the ΔPOX ΔFALDH strain under the control of Y. lipolytica-derived EXP1 promoters. Results and Discussion: The ΔPOX ΔFALDH strains with ω-CvTA successfully accumulated the corresponding C12 αω-diamines into a shaking culture medium with dodecane or dodecanol. In addition, these strains accumulated C12 ω-amino fatty acids from dodecanoic acid. With the commercially available α,ω-diacid bioprocess, this yeast biosynthesis producing medium- and longchain α,ω-diamines and ω-amino fatty acids could complete the yeast platform technology generating all medium- and long-chain aliphatic polyamide monomers, α,ω-biofunctionalized with one or both carboxylic acid and amino residues.

Engineered Escherichia coli strains as platforms for biological production of isoprene.

Volatile compounds can be produced by fermentation from genetically engineered microorganisms. Escherichia coli strains are mainly used for isoprene production owing to their higher titers; however, this has thus far been confined to only strains BL21, BL21 (DE3), Rosetta, and BW25113. Here, we tested four groups of E. coli strains for improved isoprene production, including K-12 (DH5α, BW25113, W3110, MG1655, XL1-Blue, and JM109), B [Rosetta (DE3), BL21, and BL21 (DE3)], Crooks C, and Waksman W strains. The isoprene productivity of BL21 and MG1655 was remarkably higher than that of the others in 5-L fermentation, and scale-up fermentation (300 L) of BL21 was successfully performed. This system shows potential for biobased production of fuel and volatile compounds in industrial applications.

Production of Bio-Based Isoprene by the Mevalonate Pathway Cassette in Ralstonia eutropha.

Isoprene has the potential to replace some petroleum-based chemicals and can be produced through biological systems using renewable carbon sources. Ralstonia eutropha can produce value-added compounds, including intracellular polyhydroxyalkanoate (PHA) through fatty acid and lipid metabolism. In the present study, we engineered strains of R. eutropha H16 and examined the strains for isoprene production. We optimized codons of all the genes involved in isoprene synthesis by the mevalonate pathway and manipulated the promoter regions using pLac and pJ5 elements. Our results showed that isoprene productivity was higher using the J5 promoter (1.9 ± 0.24 µg/l) than when using the lac promoter (1.5 ± 0.2 µg/l). Additionally, the use of three J5 promoters was more efficient (3.8 ± 0.18 µg/l) for isoprene production than a one-promoter system, and could be scaled up to a 5-L batch-cultivation from a T-flask culture. Although the isoprene yield obtained in our study was insufficient to meet industrial demands, our study, for the first time, shows that R. eutropha can be modified for efficient isoprene production and lays the foundation for further optimization of the fermentation process.

In-vivo half-life and hypoglycemic bioactivity of a fusion protein of exenatide and elastin-based polypeptide from recombinant Saccharomyces cerevisiae.

Exenatide (Ex) is a 39-amino acid peptide of glucagon-like peptide-1 (GLP-1) receptor agonist that was approved by the FDA in 2005 as a Type II diabetes treatment. It shows a 53% homology with GLP-1 but has an extended half-life (ca. 2.4 h) relative to GLP-1 (ca. 2-3 min). In this study, to further extend its in vivo half-life, we constructed a fusion protein (Ex-(EBP)10-6xHis) using a biocompatible and inert elastin-based polypeptide (EBP) as a fusion partner. Valine was inserted into the guest position of the pentapeptide (VPGXG), no linker sequence was inserted in between the EBPs, and (EBP)10-6xHis tag was attached to the C-terminus of exenatide. By using a recombinant Saccharomyces cerevisiae expression system, the fusion protein was expressed and secreted to the broth and purified by Ni-NTA affinity chromatography. Compared with the native exenatide, the physical half-life of the fusion protein was ca. 3.7-fold extended while approximately 72% of the in-vitro insulin secreting activity was maintained. However, the biological half-life measured by a glucose tolerance test (GTT) and the hypoglycemic test in mice was not significantly different from that of the native form. The effects of EBPylation on bioactivity and half-life of the fusion protein are similar to those of PEGylation. The result suggests that the bioactivity and half-life should be carefully balanced to obtain optimal fusion proteins. We expect that EBPylation using an optimal repeat number of EBP can be an alternative to chemical modification for therapeutic biobetters with extended half-life.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

Enhancement of L-Threonine Production by Controlling Sequential Carbon-Nitrogen Ratios during Fermentation.

Controlling the residual glucose concentration is important for improving productivity in L-threonine fermentation. In this study, we developed a procedure to automatically control the feeding quantity of glucose solution as a function of ammonia-water consumption rate. The feeding ratio (RC/N) of glucose and ammonia water was predetermined via a stoichiometric approach, on the basis of glucose-ammonia water consumption rates. In a 5-L fermenter, 102 g/l L-threonine was obtained using our glucose-ammonia water combined feeding strategy, which was then successfully applied in a 500-L fermenter (89 g/l). Therefore, we conclude that an automatic combination feeding strategy is suitable for improving L-threonine production.

A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study.

A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

Isolation and characterization of a novel ε-caprolactam-degrading microbe, Acinetobacter calcoaceticus, from industrial wastewater by chemostat-enrichment.

For the isolation of a ε-caprolactam-degrading microbe from wastewaters of a factory producing caprolactam, we applied a chemostat-enrichment technique with a selective medium containing caprolactam as sole source of carbon and nitrogen. This allowed for the isolation of a novel caprolactam-degrading microbe, identified as Acinetobacter calcoaceticus. The strain had a critical tolerance of 19 g caprolactam l(-1) in minimal medium, which is higher than any previously reported caprolactam-degrading microbe. A. calcoaceticus also decreased the caprolactam content in medium by 65 % within 72 h despite the high caprolactam content (10 g l(-1)). This study highlights the potential use of A. calcoaceticus strain for the bioremediation of recalcitrant synthetic polymers, such as caprolactam.

Media optimization of Corynebacterium glutamicum for succinate production under oxygen-deprived condition.

Corynebacterium glutamicum is one of the well-studied industrial strain that is used for the production of nucleotides and amino acids. Recently, it has also been studied as a possible producer of organic acids such as succinic acid, based on its ability to produce organic acids under an oxygen deprivation condition. In this study, we conducted the optimization of medium components for improved succinate production from C. glutamicum under an oxygen deprivation condition by Plackett-Burman design and applied a response surface methodology. A Plackett-Burman design for ten factors such as glucose, ammonium sulfate, magnesium sulfate, potassium phosphate (K2HPO4 and KH2PO4), iron sulfate, manganese sulfate, biotin, thiamine, and sodium bicarbonate was applied to evaluate the effects on succinate production. Glucose, ammonium sulfate, magnesium sulfate, and dipotassium phosphate were found to have significant influence on succinate production, and the optimal concentrations of these four factors were sequentially investigated by the response surface methodology using a Box-Behnken design. The optimal medium components obtained for achieving maximum concentration of succinic acid were as follows: glucose 10 g/l, magnesium sulfate 0.5 g/l, dipotassium phosphate (K2HPO4) 0.75 g/l, potassium dihydrogen phosphate (KH2PO4) 0.5 g/l, iron sulfate 6 mg/l, manganese sulfate 4.2 mg/l, biotin 0.2 mg/l, thiamine 0.2 mg/l, and sodium bicarbonate 100 mM. The parameters that differed from a normal BT medium were glucose changed from 40 g/l to 10 g/l, dipotassium phosphate (K2HPO4) 0.5 g/l changed to 0.75 g/l, and ammonium sulfate ((NH4)2SO4) 7 g/l changed to 0 g/l. Under these conditions, the final succinic acid concentration was 16.3 mM, which is about 1.46 fold higher than the original medium (11.1 mM) at 24 h. This work showed the improvement of succinate production by a simple change of media components deduced from sequential optimization.

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein.

BACKGROUND: The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. RESULTS: An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q(Ab)) than that of the unsorted pool. The q(Ab) was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q(Ab) in individual selected clones. CONCLUSIONS: This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q(Ab) with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

Effect of constitutively active Ras overexpression on cell growth in recombinant Chinese hamster ovary cells.

Constitutively active Ras (CA-Ras) is known to enhance cell growth through the induction of various signaling cascades including the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK signaling pathways, although the cellular response is highly dependent on the cell type. To evaluate the effect of CA-Ras overexpression on cell growth in recombinant Chinese hamster ovary (rCHO) cells, an erythropoietin (EPO)-producing rCHO cell line with regulated CA-Ras overexpression (EPO-off-CA-Ras) was established using the Tet-off system. The CA-Ras expression level in EPO-off-CA-Ras cells was tightly regulated by doxycycline addition. Although CA-Ras overexpression slightly increased the viable cell concentration during the late exponential phase, it did not increase the maximum viable cell concentration or specific growth rate to a significant degree. Unexpectedly, CA-Ras overexpression in rCHO cells led only to the enhancement in the activation of the MAPK/ERK signaling pathway and not the PI3K/Akt signaling pathway. Taken together, CA-Ras overexpression in rCHO cells did not significantly affect cell growth; it also had no critical impact on viable cell concentration or EPO production, possibly due to a failure to activate the PI3K/Akt signaling pathway.

Carbon nanotube-assisted enhancement of surface plasmon resonance signal.

We describe a method of amplifying the biosensing signal in surface plasmon resonance (SPR)-based immunoassays using an antibody-carbon nanotube (CNT) conjugate. As a model system, human erythropoietin (EPO) and human granulocyte macrophage colony-stimulating factor (GM-CSF) were detected by sandwich-type immunoassays using an SPR biosensor. For the amplification of the SPR signal, the CNT was conjugated with a polyclonal antibody, and then the conjugates were reacted with antibodies coupled with the target proteins. This amplification strategy increases the dynamic range of the immunoassays and enhances the detection sensitivity. The SPR immunoassays, combined with the CNT-assisted signal amplification method, provided a wide dynamic range over four orders of magnitude for both EPO and GM-CSF (0.1-1,000 ng/ml). The CNT amplification method is expected to realize the detection of picogram levels and a wide dynamic detection range of multiple proteins, enabling it to offer a robust analysis tool for the development of biopharmaceutical production.

Exploring the effects of carbon sources on the metabolic capacity for shikimic acid production in Escherichia coli using in silico metabolic predictions.

Effects of various industrially important carbon sources (glucose, sucrose, xylose, gluconate, and glycerol) on shikimic acid (SA) biosynthesis in Escherichia coli were investigated to gain new insight into the metabolic capability for overproducing SA. At the outset, constraints-based flux analysis using the genome-scale in silico model of E. coli was conducted to quantify the theoretical maximum SA yield. The corresponding flux distributions fueled by different carbon sources under investigation were compared with respect to theoretical yield and energy utilization, thereby identifying the indispensable pathways for achieving optimal SA production on each carbon source. Subsequently, a shikimate-kinase-deficient E. coli mutant was developed by blocking the aromatic amino acid pathway, and the production of SA on various carbon sources was experimentally examined during 5 l batch culture. As a result, the highest production rate, 1.92 mmol SA/h, was obtained when glucose was utilized as a carbon source, whereas the efficient SA production from glycerol was obtained with the highest yield, 0.21 mol SA formed per mol carbon atom of carbon source consumed. The current strain can be further improved to satisfy the theoretically achievable SA production that was predicted by in silico analysis.

Improved L-threonine production of Escherichia coli mutant by optimization of culture conditions.

L-threonine production was investigated in a minimal salt medium using L-threonine-overproducing Escherichia coli MT201, derived from E. coli K-12. It was observed that dry cell weight reached 12.5 g/l with 15.9 g/lL-threonine. To increase dry cell weight and L-threonine production, the fermentation process was optimized. When biotin was added as growth factor, L-threonine production reached 52.0 g/l from 15.9 g/l without biotin. Dry cell weight and L-threonine production were further increased by continuous feeding of the feed media with an optimized L-methionine concentration (5.0 g/l). However, high-cell-density culture caused oxygen-limited condition, which resulted in the accumulation of organic acids. To overcome this problem, oxygen-enriched air was supplied to the fermentor with the minimal salt medium. Under these optimal conditions, we achieved an L-threonine production of 80.2 g/l in the minimal salt medium.