Ginseng is a medicinal herb that requires cultivation under shade conditions, typically for 4-6 years, before harvesting. The principal components of ginseng are ginsenosides, glycosylated tetracyclic terpenes. Dammarene-type ginsenosides are classified into two groups, protopanaxadiol (PPD) and protopanaxatriol (PPT), based on their hydroxylation patterns, and further diverge to diverse ginsenosides through differential glycosylation. Three early enzymes, dammarenediol-II synthase (DS) and two P450 enzymes, protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), have been reported, but glycosyltransferases that are necessary to synthesize specific ginsenosides have not yet been fully identified. To discover glycosyltransferases responsible for ginsenoside biosynthesis, we sequenced and assembled the ginseng transcriptome de novo and characterized two UDP-glycosyltransferases (PgUGTs): PgUGT74AE2 and PgUGT94Q2. PgUGT74AE2 transfers a glucose moiety from UDP-glucose (UDP-Glc) to the C3 hydroxyl groups of PPD and compound K to form Rh2 and F2, respectively, whereas PgUGT94Q2 transfers a glucose moiety from UDP-Glc to Rh2 and F2 to form Rg3 and Rd, respectively. Introduction of the two UGT genes into yeast together with PgDS and PgPPDS resulted in the de novo production of Rg3. Our results indicate that these two UGTs are key enzymes for the synthesis of ginsenosides and provide a method for producing specific ginsenosides through yeast fermentation.

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10(-8)/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10(-8)/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.

Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.

Despite numerous approaches for the development of L-threonine-producing strains, strain development is still hampered by the intrinsic inefficiency of metabolic reactions caused by simple diffusion and random collisions of enzymes and metabolites. A scaffold system, which can promote the proximity of metabolic enzymes and increase the local concentration of intermediates, was reported to be one of the most promising solutions. Here, we report an improvement in L-threonine production in Escherichia coli using a DNA scaffold system, in which a zinc finger protein serves as an adapter for the site-specific binding of each enzyme involved in L-threonine production to a precisely ordered location on a DNA double helix to increase the proximity of enzymes and the local concentration of metabolites to maximize production. The optimized DNA scaffold system for L-threonine production significantly increased the efficiency of the threonine biosynthetic pathway in E. coli, substantially reducing the production time for L-threonine (by over 50%). In addition, this DNA scaffold system enhanced the growth rate of the host strain by reducing the intracellular concentration of toxic intermediates, such as homoserine. Our DNA scaffold system can be used as a platform technology for the construction and optimization of artificial metabolic pathways as well as for the production of many useful biomaterials.A s the building blocks of life, amino acids have long played an important role in both human and animal nutrition and health maintenance. On account of its functionality and the special features arising from chirality, this class of compounds is extremely important and of great interest for the chemical industry (1). Of the 20 standard amino acids, the 9 essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) occupy a key position, in that they are not synthesized in animals and humans but must be ingested with feed or food (2).Production methods for these essential amino acids are broadly classified into three types: extraction, chemical synthesis, and microbial fermentation. Among these methods, the microbial fermentation method is being widely applied to the industrial production of most essential amino acids, except for a few kinds of L-amino acids for which high production yields have not been achieved by fermentation (1). The advances in the industrial production of amino acids are closely connected with screening or selection of suitable putative production hosts and subsequent improvement of production strains. Previous attempts at strain improvement have relied on classical mutagenesis and screening procedures, which focused on deleting competing pathways and eliminating feedback regulations in the biosynthetic pathway (1, 3-7). The recent trend of whole-genome analysis and systems biology has begun to exert a profound effect on the strategy of strain development. The barrage of information has led to a better understanding of the architecture of cel...

g Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) in Escherichia coli led to a higher frequency of persister formation. The persister frequency of E. coli was increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-related hipA7 mutation indicated that surplus fumarate markedly elevated the E. coli persister frequency. An E. coli strain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ϳ1,000-fold more effectively than the wild-type strain in the stationary phase. It appears that SDH and FRD represent a paired system that gives rise to and maintains E. coli persisters by producing and utilizing fumarate, respectively. Bacterial persisters are phenotypic variants that are tolerant even to supralethal concentrations of multiple antibiotics (1-3). Reseeding of the persisters yields a bacterial population with a frequency of antibiotic-tolerant cells that is similar to that of the parental population (4, 5). Persisters are distinct from antibioticresistant cells because the ability to tolerate antibiotics is neither genetically determined nor inherited. Persisters showing tolerance of different classes of antibiotics are observed in most microbial species and have been implicated in chronic and recurrent infections (1). Furthermore, it is highly probable that persisters are a potential reservoir for the development of drug resistance in pathogenic bacteria (6, 7).Despite the discovery of bacterial persisters more than 70 years ago (4), the mechanisms that underlie noninheritable persistence phenotypes remain unclear. Various researchers recently identified a number of genes and pathways that lead to persister formation or survival upon antibiotic treatments. These include toxinantitoxin (TA) modules, a stringent response, phosphate metabolism, alternative energy production, and antioxidative defense (8-13). Because nongrowing or slow-growing bacteria are less sensitive to antibiotics, dormancy has been proposed to be the mechanism of last resort in many of these persistence studies. Thus, many recent mechanistic studies have focused on how bacterial cells reach the dormant state (8-15). Nonetheless, the prevailing hypothesis that persisters might survive solely because of dormancy is being challenged. A lack of significant growth or metabolic activity does not guarantee persistence, and dormancy is neither necessary nor sufficient fo...

Minor ginsenosides, such as compound K, Rg3(S), which can be produced by deglycosylation of ginsenosides Rb1, showed strong anti-cancer effects. However, the anticancer effects of gypenoside LXXV, which is one of the deglycosylated shapes of ginsenoside Rb1, is still unknown due to the rarity of its content in plants. Here, we cloned and characterized a novel ginsenoside-transforming β-glucosidase (BglG167b) derived from Microbacterium sp. Gsoil 167 which can efficiently hydrolyze gypenoside XVII into gypenoside LXXV, and applied it to the production of gypenoside LXXV at the gram-scale with high specificity. In addition, the anti-cancer activity of gypenoside LXXV was investigated against three cancer cell lines (HeLa, B16, and MDA-MB231) in vitro. Gypenoside LXXV significantly reduced cell viability, displaying an enhanced anti-cancer effect compared to gypenoside XVII and Rb1. Taken together, this enzymatic method would be useful in the preparation of gypenoside LXXV for the functional food and pharmaceutical industries.

Yeasts are very useful microorganisms that are used in many industrial fermentation processes such as food and alcohol production. Microbial contamination of such processes is inevitable, since most of the fermentation substrates are not sterile. Contamination can cause a reduction of the final product concentration and render industrial yeast strains unable to be reused. Alternative approaches to controlling contamination, including the use of antibiotics, have been developed and proposed as solutions. However, more efficient and industry-friendly approaches are needed for use in industrial applications. This review covers: (i) general information about industrial uses of yeast fermentation, (ii) microbial contamination and its effects on yeast fermentation, and (iii) currently used and suggested approaches/strategies for controlling microbial contamination at the industrial and/or laboratory scale.

A point mutation (E115K) resulting in slower growth of Escherichia coli DH5␣ and XL1-Blue in minimal media was identified in the purB gene, coding for adenylosuccinate lyase (ASL), through complementation with an E. coli K-12 genomic library and serial subcultures. Chromosomal modification reversing the mutation to the wild type restored growth phenotypes in minimal media.The Escherichia coli DH5␣ strain possesses many beneficial genotypes (recA, deoR, gyrA, and endA1) and has been widely used for many purposes, such as gene cloning and protein production (5). However, E. coli DH5␣ also exhibits inferior growth phenotypes, especially in minimal media, compared to other E. coli strains. As such, the utilization of this bacterium has been limited to the laboratory despite its numerous advantages. We can assume that these inferior growth phenotypes have resulted from unknown accumulated mutations during the strain development process (5). Some of those mutations, which might impact growth in minimal media, have been characterized, including the phenotypes for thiamine requirement and relaxed amino acid synthesis (5). Still, there may be other uncharacterized mutations whose interactions hamper the growth of E. coli DH5␣ in minimal media.Based on successful identifications (6, 7) of gene targets for metabolic engineering (3), we performed serial subcultures of E. coli DH5␣ transformants with an E. coli K-12 genomic library based on a multicopy plasmid (9) to isolate genes that improve growth phenotypes in minimal media. The M9 minimal medium and R medium (11) were chosen for enrichment experiments because of their popular use in metabolic engineering (1, 2, 7) and in high-cell-density fermentation (8, 10, 11). After 11 serial transfers of the transformants in the M9 medium, and 27 transfers in the R medium, cultured cells were diluted and plated onto LB agar for single-colony isolation. Although more than 10 colonies were picked, only three distinctive plasmids, containing different inserts, were isolated from the transformants enriched in M9 medium. In the case of R medium enrichment, all isolated plasmids were identical. Sequencing of the isolated plasmids revealed the exact genome coordinates of each insert. A diagram of the inserts in the context of the E. coli genome sequence is shown in Fig. 1. Interestingly, all of the isolated plasmids contained similar regions of genomic DNA. mnmA (tRNA 5-methylaminomethyl-2-thiouridylate-methyltransferase), purB (adenylosuccinate lyase), and hflD (lysogenization regulator) were the annotated genes in the overlapping region among distinctive isolated fragments. However, since the N-terminal portions of mnmA and hflD were truncated in some of the inserts, we selected only the M3 and R1 plasmids for further experimentation. These two plasmids were retransformed into E. coli DH5␣ for confirmation of their beneficial effects on growth of E. coli in minimal media. The newly transformed strains showed growth phenotypes almost identical to those of the previously isolated transfor...