Growth regulators and their reflection on different hop genotypes cultivated under in vitro conditions / Reguladores de crescimento e seu reflexo em diferentes genótipos de lúpulo cultivados in vitro

Hops is a new culture in Brazil. Tissue culture can be an important technique for rapid hop propagation. This paper aims to characterize responses from different genotypes under different growth regulators through the interrelationship of response variables important to hop in vitro growth. Three genotypes were cultivated in six culture media with different combinations of growth regulators, BAP (6-benzylaminopurine), IAA (3-indolacetic acid) and GA3 (gibberellic acid). The means were compared by orthogonal contrasts and the interrelationship of the response variables was performed by path analysis. American genotypes showed favorable root development under the BAP + IAA combination, while the use of IAA improved shoot development. The origin of genotypes was important for defining the best protocol for in vitro cultivation. The path coefficient showed that the variable number of shoots has stronger direct effect on the number of nodal segments. Additionally, in tissue culture assays, the use of a covariable and proper error distribution significantly increased experimental accuracy.

O lúpulo é uma nova cultura no Brasil. A cultura de tecidos pode ser uma técnica importante para a propagação rápida do lúpulo. Este artigo tem como objetivo caracterizar respostas de diferentes genótipos sob diferentes reguladores de crescimento através da inter-relação de variáveis de resposta importantes para o crescimento in vitro. Três genótipos foram cultivados em seis meios de cultura com diferentes combinações de reguladores de crescimento, BAP (6-benzilaminopurina), AIA (ácido 3-indolacético) e GA3 (ácido giberélico). As médias foram comparadas por contrastes ortogonais e a inter-relação das variáveis de resposta foi realizada por análise de trilha. Os genótipos americanos apresentaram desenvolvimento radicular favorável sob a combinação BAP + AIA, enquanto o uso do AIA melhorou o desenvolvimento da parte aérea. A origem dos genótipos foi importante para definir o melhor protocolo para o cultivo in vitro. O coeficiente de trilha mostrou que a variável número de brotos tem um efeito direto mais forte no número de segmentos nodais. Adicionalmente, em experimentos com cultura de tecidos, o uso de uma covariável e distribuição de erro adequada aumentou significativamente a precisão experimental.

Growth curve of Nile tilapia from different families of the AquaAmérica variety / Curva de crescimento de tilápia-do-Nilo de diferentes famílias da variedade AquaAmérica

Selection can affect growth, changing performance and asymptotic values. However, there is little information about the growth of families in fish breeding programs. The aim of this study was to evaluate the performance and growth of families of Nile tilapia AquaAmérica. Twenty AquaAmérica families cultivated in a net cage (13.5 m3) for 181 days were evaluated. The nonlinear Gompertz regression model was fitted to the data by the weighted least squares method, taking the inverse of the variance of weight in different families and at different ages as the weighting variable. The model was adjusted to describe the growth in weight and morphometric characteristics. Two families showed highest (P<0.05) weights at both 133 days (family AA10: 743.2 g; family AA16: 741.2 g) and 181 days (family AA10: 1,422.1 g; family AA16: 1,393.4 g) of the experiment. In both experimental periods, the males showed a heavier weight, with the greatest contrast between the sexes occurring at 181 days. The analysis of the three most contrasting families (AA1, AA9 and AA14) showed that the asymptotic value for weight was higher (P<0.05) in family AA9 (3,926.3 g) than in family AA14 (3,251.6 g), but specific growth rate and age at the inflection point did not differ significantly between families. In conclusion, two of the 20 families were superior; males exhibited a greater growth, mainly in the period of 181 days; and the growth curve differed between the families, especially for asymptotic weight.

A seleção pode impactar a forma de crescimento, mudando o desempenho e os valores assintóticos. No entanto, existem poucas informações sobre o crescimento das famílias em programas de criação de peixes. O objetivo deste estudo foi examinar o desempenho e as curvas de crescimento de famílias de tilápia-do-Nilo AquaAmérica. Foram avaliadas 20 famílias AquaAmérica cultivadas em tanques-rede (13,5 m3) por 181 dias. O modelo de regressão não linear de Gompertz foi ajustado aos dados pelo método dos mínimos quadrados ponderados, tomando o inverso da variância do peso nas diferentes famílias e nas diferentes idades como variável de ponderação. O modelo foi ajustado para descrever o crescimento em peso e características morfométricas. Duas famílias apresentaram pesos maiores (P <0,05) em 133 dias (família AA10: 743,2 g; família AA16: 741,2 g) e 181 dias (família AA10: 1422,1 g; família AA16: 1393,4 g) de experimento em relação a outras famílias. Em ambos os períodos experimentais, os machos apresentaram maior peso, com maior contraste entre os sexos ocorrendo aos 181 dias. A análise das três famílias mais contrastantes (AA1, AA9 e AA14) mostrou que o valor assintótico para o peso foi maior (P <0,05) na família AA9 (3926,3 g) do que na família AA14 (3251,6 g), mas a taxa de crescimento específica e a idade no ponto de inflexão não diferiu significativamente entre as famílias. Em conclusão, duas das 20 famílias eram muito superiores; machos exibiram um maior crescimento, principalmente no período de 181 dias; e a curva de crescimento diferiu entre as famílias, principalmente quanto ao peso assintótico.

Genetic parameters and genetic trends of conformation and management traits in Dairy Gir cattle / [Parâmetros genéticos e tendências genéticas de características de conformação e manejo de bovinos Gir Leiteiro]

The objective of this study was to estimate genetic parameters and genetic trends of different conformation and management traits regularly measured within the context of the National Dairy Gir Breeding Program (PNMGL). The estimation of genetic and residual variances for each trait was performed using average information restricted maximum likelihood (AI-REML) procedure in AIREMLF90 program software. The population was divided into three subpopulations constituted by measured females (with phenotype records), all females, and males. Linear regressions were applied for each trait, considering two periods of birth (1st period: 1938-1996; 2nd period: 1997-2012). The estimated heritability of conformation and management traits varied from 0.01 to 0.53, denoting a perspective of genetic improvement through selection and corrective matings for purebred Dairy Gir populations. The average genetic changes in conformation and management traits were, in general, variable and inexpressive, showing that the selection of Dairy Gir may have had been directed essentially to increase milk yield. The analysis of the two periods of birth indicated that some linear traits present progress (although inexpressive) in the 2nd period (more recent period).(AU)

O objetivo deste estudo foi estimar os parâmetros genéticos e as tendências genéticas para diferentes características de conformação e manejo de animais puros da raça Gir Leiteiro, pertencentes ao Programa Nacional de Melhoramento do Gir Leiteiro (PNMGL). A estimativa das variâncias genéticas e residuais para cada característica foi realizada usando-se o procedimento de máxima verossimilhança restrita (AI-REML), por meio do programa AIREMLF90. A população foi dividida em três subpopulações, constituídas por fêmeas mensuradas (com registros de fenótipo), todas fêmeas e machos. As regressões lineares para cada característica foram ainda divididas em dois períodos de anos de nascimento (1º período: 1938 a 1996; 2º período: 1997 a 2012). As herdabilidades estimadas variaram de 0,01 a 0,53 para as características de conformação e manejo, possibilitando a perspectiva de melhoramento mediante seleção e acasalamentos corretivos na população pura da raça Gir Leiteiro. As mudanças genéticas nas características conformação e manejo foram, em geral, variáveis e inexpressivas, sugerindo que a seleção no Gir Leiteiro possa ter sido direcionada essencialmente para maior produção de leite. Ao serem observados os dois períodos distintos de anos de nascimento, infere-se que algumas características lineares apresentaram progresso (embora inexpressivo) no 2º período analisado.(AU)

Genetic tools for the development of recombinant lactic acid bacteria.

Lactic acid bacteria (LAB) are a phylogenetically diverse group with the ability to convert soluble carbohydrates into lactic acid. Many LAB have a long history of safe use in fermented foods and are recognized as food-grade microorganisms. LAB are also natural inhabitants of the human intestinal tract and have beneficial effects on health. Considering these properties, LAB have potential applications as biotherapeutic vehicles to delivery cytokines, antigens and other medicinal molecules. In this review, we summarize the development of, and advances in, genome manipulation techniques for engineering LAB and the expected future development of such genetic tools. These methods are crucial for us to maximize the value of LAB. We also discuss applications of the genome-editing tools in enhancing probiotic characteristics and therapeutic functionalities of LAB.

[The crispr / cas9 techniques applied to human genetic enhancement: a biotechnological, anthropological-philosophical and legal dialogue]. / Las técnicas crispr/cas9 aplicadas al mejoramiento genético humano: un diálogo biotecnológico, antropológico-filosófico y jurídico.

The CRISPR editing method is revolutionary. This technique opens the possibility of countless operations in the genome of living beings. However, the risks are high and, in some cases, unpredictable. Therefore, based on an anthropology that recognizes the human person with an inherent dignity that includes the body, this article intends to propose bases for a regulation capable of facing the challenge of CRISPR, especially, given the possibility of confusing its therapeutic resource with the eugenics, also before the imminent risk of unleashing unforeseen consequences such as mutations, malformations and side effects that could be devastating for human life.

Reproductive traits in different Nile tilapia genetic groups / [Características reprodutivas em diferentes grupos genéticos de tilápia do Nilo]

The aim of this study was to evaluate the reproductive traits of the non-inbred and inbred AquaAmérica, GIFT and AquaAmérica × GIFTgenetic groups. Six fish from each genetic group were used (2 females:1 male). Females were examined for the presence of eggs in their mouth at every four days, for 12 weeks. Reproduction occurred in all genetic groups (GIFT: 100%; non-inbred AquaAmérica and AquaAmérica ×GIFT: 75%; inbred AquaAmérica: 50%). Female weight, female standard length, total spawning weight, absolute fecundity, relative fecundity, spawn index and hatching rate did not differ significantly between the genetic groups. However, the non-inbred AquaAmérica variety showed lower values (P<0.05) for egg diameter (2.4mm) and egg weight (4.2mg) and higher values (P<0.05) for relative number of eggs (247.6 eggs/g of egg) than GIFT (egg diameter: 2.8mm; egg weight: 5.7mg; relative number of eggs: 175.4 eggs/g of egg) and AquaAmérica ×GIFT (egg diameter: 2.8mm; egg weight: 5.9mg; relative number of eggs: 168.8 eggs/g of egg). In conclusion, the non-inbred AquaAmérica variety produces smaller, lighter eggs but a higher relative number of eggs than the GIFT variety and the AquaAmérica ×GIFT cross; and inbreeding negatively affects spawning rate.(AU)

O objetivo deste estudo foi avaliar as características reprodutivas dos grupos genéticos AquaAmérica não endogâmicos e endogâmicos, GIFT e AquaAmérica × GIFT. Foram utilizados seis peixes de cada grupo genético (duas fêmeas:um macho). As fêmeas foram examinadas quanto à presença de ovos na boca a cada quatro dias, durante 12 semanas. A reprodução ocorreu em todos os grupos genéticos (GIFT: 100%; AquaAmérica não endogâmica e AquaAmérica × GIFT: 75%; AquaAmérica endogâmica: 50%). Peso e comprimento padrão de fêmea, peso total de desova, fecundidade absoluta, fecundidade relativa, índice de desova e taxa de eclosão não diferiram significativamente entre os grupos genéticos. Entretanto, a variedade não endogâmica da AquaAmérica apresentou valores mais baixos (P<0,05) para diâmetro do ovo (2,4mm) e peso do ovo (4,2mg) e maiores valores (P<0,05) para número relativo de ovos (247,6 ovos/g de ovo ) que GIFT (diâmetro do ovo: 2,8mm; peso do ovo: 5,7mg; número relativo de ovos: 175,4 ovos/g de ovo) e AquaAmérica × GIFT (diâmetro do ovo: 2,8mm; peso do ovo: 5,9mg; número relativo de ovos: 168,8 ovos/g de ovo). Em conclusão, a variedade AquaAmérica não endogâmica produz ovos menores e mais leves, mas um número relativo maior de ovos que a variedade GIFT e o cruzamento AquaAmérica × GIFT; a consanguinidade afeta negativamente a taxa de desova.(AU)

Enhancer-Driven Gene Expression (EDGE) enables the generation of cell type specific tools for the analysis of neural circuits.

As in all circuits, fully understanding how neural circuits operate requires the ability to specifically manipulate individual circuit elements, i.e. particular neuronal cell types. While recent years saw the development of molecular genetic tools allowing one to control and monitor neuronal activity, progress is limited by the ability to express such transgenes specifically enough. This goal is complicated by the fact that we are only beginning to understand how many cell types exist in the mammalian brain. Obtaining neuronal cell type-specific expression requires co-opting the genetic machinery which specifies their striking diversity, typically done by making transgenic animals using promoters expressing in neurons. However, while the vast majority of genes express in the brain, they almost always express in multiple cell types, meaning native promoters are not specific enough. We have recently taken a new approach to increase the specificity of transgene expression based upon identifying the distal cis-regulatory genomic elements (i.e. enhancers) uniquely active in a brain region and combining them with a heterologous minimal promoter. Termed Enhancer-Driven Gene Expression (EDGE), it allows for the generation of transgenic animals targeting the cell types of any brain region with far greater specificity than can be obtained with native promoters. Moreover, their small size allows for the generation of cell-specific viral vectors, conceivably enabling circuit-specific manipulations to any species.

Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis.

CotA-laccases are potential enzymes that are widely used in decolorization of dyes and degradation of toxic substances. In this study, a novel CotA-laccase gene from Bacillus pumilus W3 was applied for rational design. After a series of site-directed genetic mutations, the mutant S208G/F227A showed a 5.1-fold higher catalytic efficiency (kcat/Km) than the wild-type CotA-laccase did. The optimal pH of S208G/F227A was 3.5 with ABTS as substrate. The residual activity of mutant S208G/F227A was more than 80% after incubated for 10 h at pH 7-11. Mutant S208G/F227A showed optimal temperature at 80°C with ABTS as substrate. The thermal stability of mutant laccase S208G/F227A was lower than that of wild-type CotA-laccase. This study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis.

Genetic enhancement for semi-dwarf and bacterial blight resistance with enhanced grain quality characteristics in traditional Basmati rice through marker-assisted selection.

Ranbir Basmati is one of the traditional Basmati varieties of India and of the most popular traditional Basmati variety grown in Jammu's region (State of Jammu & Kashmir). It is a tall and short-duration variety with strong aroma and excellent cooking quality. However, it is susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv oryzae (Xoo) and prone to lodging. In this study, semi-dwarf (sd1) and BB resistance genes (Xa21 and xa13) were introgressed into Ranbir Basmati using marker-assisted backcross breeding (MABB) scheme. A high-yielding PAU148 carrying Xa21, xa13 and sd1 genes was used as a donor parent. On each generation target, genes were selected, while polymorphic SSR markers were used to select plants having maximum recovery of the recurrent genome. The maximum genome recovery of Ranbir Basmati in BC2F2 was 86.9% in introgressed line SBTIL121. The genotypes carrying resistant genes exhibited very high levels of tolerance against BB disease along with good Basmati rice grain quality traits. The agronomic traits of introgressed lines evaluated in the field and the laboratory showed that most of the agro-morphological traits were similar or superior to Ranbir Basmati. The identified lines can be further evaluated and released as Improved Ranbir Basmati variety.

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis.

For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine-tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5'- and 3'-end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.

Application of genome-editing systems to enhance available pig resources for agriculture and biomedicine.

Traditionally, genetic engineering in the pig was a challenging task. Genetic engineering of somatic cells followed by somatic cell nuclear transfer (SCNT) could produce genetically engineered (GE) pigs carrying site-specific modifications. However, due to difficulties in engineering the genome of somatic cells and developmental defects associated with SCNT, a limited number of GE pig models were reported. Recent developments in genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system, have markedly changed the effort and time required to produce GE pig models. The frequency of genetic engineering in somatic cells is now practical. In addition, SCNT is no longer essential in producing GE pigs carrying site-specific modifications, because direct injection of genome-editing systems into developing embryos introduces targeted modifications. To date, the CRISPR/Cas9 system is the most convenient, cost-effective, timely and commonly used genome-editing technology. Several applicable biomedical and agricultural pig models have been generated using the CRISPR/Cas9 system. Although the efficiency of genetic engineering has been markedly enhanced with the use of genome-editing systems, improvements are still needed to optimally use the emerging technology. Current and future advances in genome-editing strategies will have a monumental effect on pig models used in agriculture and biomedicine.

Combination of biomaterial transplantation and genetic enhancement of intrinsic growth capacities to promote CNS axon regeneration after spinal cord injury.

The inhibitory environment that surrounds the lesion site and the lack of intrinsic regenerative capacity of the adult mammalian central nervous system (CNS) impede the regrowth of injured axons and thereby the reestablishment of neural circuits required for functional recovery after spinal cord injuries (SCI). To circumvent these barriers, biomaterial scaffolds are applied to bridge the lesion gaps for the regrowing axons to follow, and, often by combining stem cell transplantation, to enable the local environment in the growth-supportive direction. Manipulations, such as the modulation of PTEN/mTOR pathways, can also enhance intrinsic CNS axon regrowth after injury. Given the complex pathophysiology of SCI, combining biomaterial scaffolds and genetic manipulation may provide synergistic effects and promote maximal axonal regrowth. Future directions will primarily focus on the translatability of these approaches and promote therapeutic avenues toward the functional rehabilitation of patients with SCIs.

Overexpression of germin-like protein GmGLP10 enhances resistance to Sclerotinia sclerotiorum in transgenic tobacco.

Germin-like proteins (GLPs) are ubiquitous water-soluble glycoproteins that are located in the extracellular matrix. These proteins have been reported to play vital roles in diverse biological processes. In the present study, a GLP in soybean (Glycine max L. Merr.), GmGLP10, was characterized. Sequence analysis revealed that the GmGLP10 gene (GenBank Accession Number EU916258) encodes a 213-amino acid (aa) protein, which contains a N-terminal signal peptide at 1-22 aa and is highly homologous to the members of the GER2 subfamily. GmGLP10 was highly expressed in the leaves, but very faint in the roots. The expression of GmGLP10 was induced by methyl jasmonate (MeJA), ethylene (ET), salicylic acid (SA), oxalate acid (OA), and the infection of Sclerotinia sclerotiorum. Overexpression of GmGLP10 in transgenic tobacco significantly enhanced tolerance to OA and S. sclerotiorum infection. Moreover, higher levels of H2O2 and the upregulated expression of a set of plant defense-related genes and HR (hypersensitive response)-associated genes were detected in the transgenic plants. These results suggest that GmGLP10 functions as a positive regulator of resistance to S. sclerotiorum.

Comparison of two hyoscyamine 6ß-hydroxylases in engineering scopolamine biosynthesis in root cultures of Scopolia lurida.

Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6ß-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.

Molecular cloning and characterization of the glutathione reductase gene from Stipa purpurea.

Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.

Construction of a high-yield dengue virus by replacing nonstructural proteins 3-4B without increasing virulence.

Producing virus at high yield is critically important for development of whole virion inactivated vaccines or live attenuated vaccines. Most dengue virus (DENV) clinical isolates, however, replicate at low levels in cultured cells, which limits their use for vaccine development. The present study examined differences between low-replicating DENV clinical isolates and high-replicating laboratory strains with the aim of engineering high-yield DENV clinical isolates. Construction of a series of recombinant chimeric viruses derived from a high-replicating laboratory DENV type 4 (DENV-4) H241 strain and a clinical isolate revealed that the NS3-NS4B region of H241 conferred a replication advantage in cultured cells. Furthermore, northern blot analysis revealed that this advantage was due to more efficient synthesis of viral RNA. Importantly, replacement of the NS3-NS4B region of H241 did not increase virulence in mice, suggesting that viral production can be increased safely. This study provided information that will facilitate engineering of safe and high-yield viruses that can be used for vaccine development.

Producing a glycosylating Escherichia coli cell factory: The placement of the bacterial oligosaccharyl transferase pglB onto the genome.

Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%.

Identification and manipulation of a novel locus to improve cell tolerance to short-chain alcohols in Escherichia coli.

Escherichia coli KO11 is a popular ethanologenic strain, but is more sensitive to ethanol than other producers. Here, an ethanol-tolerant mutant EM was isolated from ultraviolet mutagenesis library of KO11. Comparative genomic analysis added by piecewise knockout strategy and complementation assay revealed EKO11_3023 (espA) within the 36.6-kb deletion from KO11 was the only locus responsible for ethanol sensitivity. Interestingly, when espA was deleted in strain W (the parent strain of KO11), ethanol tolerance was dramatically elevated to the level of espA-free hosts [e.g., MG1655 and BL21(DE3)]. And overexpression of espA in strains MG1655 and BL21(DE3) led to significantly enhanced ethanol sensitivity. In addition to ethanol, deletion of espA also improved cell tolerance to other short-chain (C2-C4) alcohols, including methanol, isopropanol, n-butanol, isobutanol and 2-butanol. Therefore, espA was responsible for short-chain alcohol sensitivity of W-strains compared to other cells, which provides a potential engineering target for alcohols production.

Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells.

One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications.