Genetic engineering approaches to improve bioethanol production from maize

Biofuels such as bioethanol are becoming a viable alternative to fossil fuels. Utilizing agricultural biomass for the production of biofuel has drawn much interest in many science and engineering disciplines. As one of the major crops, maize offers promise in this regard. Compared to other crops with biofuel potential, maize can provide both starch (seed) and cellulosic (stover) material for bioethanol production. However, the combination of food, feed and fuel in one crop, although appealing, raises concerns related to the land delineation and distribution of maize grown for energy versus food and feed. To avoid this dilemma, the conversion of maize biomass into bioethanol must be improved. Conventional breeding, molecular marker assisted breeding and genetic engineering have already had, and will continue to have, important roles in maize improvement. The rapidly expanding information from genomics and genetics combined with improved genetic engineering technologies offer a wide range of possibilities for enhanced bioethanol production from maize.     

Gene replacement through homologous recombination in Mycobacterium intracellulare.

Mycobacterium intracellulare is a slow-growing pathogenic mycobacterium closely related to Mycobacterium avium. In contrast to Mycobacterium tuberculosis and Mycobacterium bovis BCG, M. intracellulare has received little attention as a model species for studies of mycobacterial molecular biology and genetics. This study shows that M. intracellulare 1403 (ATCC 35761) can be transformed by electroporation with high frequencies (up to 10(6) transformants per microgram of DNA), using plasmids pYT937 and pMH94 as replicative and integrative vectors, respectively. We also describe an experimental system that we used to study DNA recombination in M. intracellulare. First, an integrative plasmid was introduced into M. intracellulare 1403. A nonreplicative, nonintegrative plasmid having homology with the integrated plasmid was then introduced, and the resultant recombinants were analyzed to distinguish between events of homologous and illegitimate recombination. No illegitimate recombination occurred; in all recombinants, a single crossover between homologous regions of the two plasmids was noted. During subsequent growth of a recombinant clone, a spontaneous deletion occurred that resulted in a gene replacement on the chromosome of M. intracellulare 1403. The ability to construct site-specific mutations in M. intracellulare will provide novel insights into the biology of slow-growing mycobacteria.     

Disruption of Thermus thermophilus genes by homologous recombination using a thermostable kanamycin-resistant marker

Genes of an extremely thermophilic bacterium, Thermus thermophilus, were disrupted by homologous recombination using a recently developed, thermostable kanamycin-resistant marker. First, the trpE gene was disrupted with various constructions of DNA. The transformation efficiency was exponentially increased as the length of the homologous regions flanking the marker gene increased above the minimum length (200-300 bp). We then disrupted five genes of the nucleotide excision repair system and examined their phenotypes. The convenience and high reliability of this method should prompt its application to the high-throughput systematic disruption of the genes of this thermophilic bacterium.     

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.     

Disruption of the genomic imprint in trans with homologous recombination at Snrpn in ES cells

In gene targeting studies of the Prader-Willi syndrome (PWS)/Angelman syndrome (AS) domain in mouse ES cells, we recovered only recombinants with the paternal allele for constructs at exons 2 or 3 of the imprinted, maternally silenced Snurf-Snrpn gene. These sites lie close to the imprinting center (IC) for this domain. In contrast, recombinants for Ube3a within the same imprinted domain were recovered with equal frequency on the maternal and paternal alleles. In addition, gene targeting of the paternal allele for Snurf-Snrpn resulted in partial or complete demethylation in trans with activation of expression for the previously silenced maternal allele. The imprint switching of the maternal allele in trans is not readily explained by competition for trans-acting factors and adds to a growing body of evidence indicating homologous association of oppositely imprinted chromatin domains in somatic mammalian cells.     

基因工程在β-内酰胺药物研制上的应用

本文综合国内外有关文献介绍了青酶素Ｇ酰化酶的改造,异青霉素Ｎ合成酶和头孢菌素Ｃ水解酶的克隆,以及细菌血红蛋白基因克隆进丝状真菌。比较了化学法和两步酶法转化ｃｐｃ（头孢菌素Ｃ）成７－ＡＣＡ的优缺点。还讨论了基因工程β－内酰胺抗生素研制的发展方向。     

Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant

An adhE, ldhA double mutant Escherichia coli strain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl-CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphere of CO(2) yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsG inactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICB(glu) can be rediverted toward OAA favoring succinate production.     

Strain differences in the immune response of mice to homologous sperm-specific lactate dehydrogenase (LDH-C4).

The sperm-specific isozyme of murine lactate dehydrogenase (LDH-C4) was injected into female mice of various strains. Two regulatory phenotypes characterize the resultant immunity to LDH-C4: one is manifested by high, intermediate or low levels of response, the other by the immediate or delayed maturation of peak titer. The response of several strains can be classified as high (SWR, SJL, BABL/c, C3H/He) and intermediate to low (A, CBA, DBA/2, DBA/1, C57BL/6) according to the level of antibody production and cell mediated immunity. BALB/c, SJL and SWR strains are immediate responders while DBA/2 and C3H/He mice are clearly delayed responders. Maturation and magnitude of response do not appear to be related. Both the antibody and cell mediated responses are T-dependent, but are not obviously associated with Ig allotype or H-2 regulation.     

Stimulation of homologous recombination through targeted cleavage by chimeric nucleases

Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.     

Utilization of citrate and lactate through a lactate dehydrogenase and ATP-regulated pathway in boar spermatozoa

Incubation of boar spermatozoa in Krebs-Ringer-Henseleit medium with either 10 mM lactate or 10 mM citrate induced a fast and robust increase in the intracellular levels of ATP in both cases, which reached a peak after 30 sec of incubation. Utilization of both citrate and lactate resulted in the export of CO(2) to the extracellular medium, indicating that both substrates were metabolized through the Krebs cycle. Incubation with citrate resulted in the generation of extracellular lactate, which was inhibited in the presence of phenylacetic acid. This indicates that lactate is produced through the pyruvate carboxylase step. In addition, there was also a significant increase in tyrosine phosphorylation induced by both citrate and lactate. Boar sperm has a sperm-specific isoform of lactate dehydrogenase (LDH), mainly located in the principal piece of the tail. Kinetic studies showed that boar sperm has at least two distinct LDH activities. The major activity (with an estimated Km of 0.51 mM) was located in the supernatants of sperm extracts. The minor LDH activity (with an estimated Km of 5.9 mM) was associated with the nonsoluble fraction of sperm extracts. Our results indicate that boar sperm efficiently metabolizes citrate and lactate through a metabolic pathway regulated by LDH.     

Construction of squalene-accumulatingSaccharomyces cerevisiae mutants by gene disruption through homologous recombination

Saccharomyces cerevisiae synthesizes ergosterol via squalene, but squalene is hardly detected in aerobically grown cells. To obtain a stable squalene-accumulating yeast strain, we attempted to disrupt a gene required in the conversion of squalene to ergosterol, by homologous recombination with a short piece of the gene fragment conjugated with an integration plasmid vector carrying theLEU2 gene. Two mutants that required ergosterol at least for fast growth were isolated. In an aerobic cultivation and with ergosterol supplementation, the two mutants accumulated squalene up to 5 mg/g dry cells. Southern hybridization analysis indicated that both mutants had acquired the vector DNA integrated in the same gene, or nearby genes, on chromosome 12.     

Disruption of the Pfg27 locus by homologous recombination leads to loss of the sexual phenotype in P. falciparum.

Transmission of malaria depends upon the differentiation and development of the sexual stages of the parasite. In Plasmodium falciparum, it is a complex, multistage process, involving the expression of a large number of sexual stage-specific proteins. Pfg27 is one such protein, abundantly expressed at the onset of gametocytogenesis. We report successful disruption of the Pfg27 locus using homologous recombination and show that it is essential for the maintenance of the sexual phenotype. Transfectants lacking Pfg27 abort early in sexual development, resulting in vacuolated, highly disarranged, and disintegrating parasites. This suggests a critical role for Pfg27 in the sexual development of the parasite.     

Keeping homologous recombination in check

Pathway choice is a critical event in the repair of DNA double-strand breaks. In a recent paper published in Nature, Orthwein et al. define a mechanism by which homologous recombination is controlled in G1 cells to favor non-homologous end joining.Homologous recombination (HR) is an essential process that produces genetic variation during meiosis and protects the genome during mitotic cell division¹. Inherited mutations in various HR factors, including the BRCA1, BRCA2 and PALB2 tumor suppressors, predispose to the development of cancer. Although HR is generally beneficial for maintaining genome integrity, HR events between homologous chromosomes can also be deleterious and lead to loss of genetic information. HR is therefore suppressed during G1 phase and in non-dividing cells, yet, the exact mechanism behind this phenomenon has remained elusive. New work from the laboratory of Daniel Durocher describes a mechanism that is both necessary and sufficient for the suppression of HR in G1 cells².DNA double-strand breaks (DSBs) are one of the most dangerous types of DNA lesion and need to be eliminated to prevent the accumulation of mutations. DSB repair is carried out by two main pathways, HR and non-homologous end joining (NHEJ)¹. Whereas NHEJ is an error-prone process that simply fuses the two broken ends together, HR is essentially error-free as it uses the genetically identical sister chromatid as a template for repair. Due to the cell cycle-dependent availability of sister chromatids, HR is restricted to the S and G2 phases of the cell cycle.In the HR repair pathway, the DSB ends are first resected to produce extended single-stranded DNA (ssDNA) tails by the coordinated actions of a series of helicase and nuclease activities (e.g., MRN, CtIP and EXO1)¹. CtIP plays a particularly important role in regulating resection, which is mediated through its interaction with BRCA1³. In the following cascade of events, BRCA1 interacts directly with the BRCA2-PALB2 complex, which in turn is recruited to the ssDNA where it acts as a chaperone that stimulates the formation of RAD51 nucleoprotein filaments that drive homology-directed HR repair to restore the integrity of the DNA^4,5.Whereas most HR events take place between the newly replicated sister chromatids, recombination between homologous chromosomes can result in loss of heterozygosity, a potentially mutagenic event that can lead to the inactivation of tumor suppressors or activation of oncogenes. HR must therefore be tightly regulated and effectively suppressed in G1 phase, at the time when only homologous chromosomes are available for repair. At such times, NHEJ is the favored mechanism for DSB repair.A number of mechanisms regulate HR to a specific phase of the cell cycle. For example, CtIP is activated for interaction with BRCA1 by CDK-dependent phosphorylation, which occurs in the S and G2 phases of the cell cycle. Conversely, HR is suppressed in G1 phase by the pro-NHEJ factors 53BP1⁶, RIF1⁷ and REV7⁸, which impair the recruitment of BRCA1 and thereby inhibit DNA end resection. Consequently, disruption of 53BP1 leads to the recruitment of BRCA1 to DSBs in G1 phase. In the recent Nature paper from Durocher''s laboratory, Orthwein et al.² discovered that although BRCA1 is localized to DSBs during G1 phase in 53BP1-deficient cells, it fails to recruit the BRCA2-PALB2 complex, which is consistent with the lack of HR activity in these cells.Through immunoprecipitation experiments Orthwein et al. showed that while BRCA2 and PALB2 interact throughout the cell cycle, BRCA1 and PALB2 only interact efficiently in S phase, suggesting that there might be a mechanism that restricts their interaction to S and G2 phases, while also blocking it in G1 phase. The region of PALB2 that is responsible for its cell cycle-regulated interaction with BRCA1 was localized to its N-terminal domain, which corresponds to a known interaction site for KEAP1, a substrate adaptor for the CUL3-RING (CRL3) ubiquitin ligase. Remarkably, they found that deletion of the KEAP1 gene using CRISPR-Cas9 technology restored the BRCA1-PALB2 interaction in G1 cells, and led to the recruitment of BRCA2-PALB2 to sites of DNA damage in 53BP1-deficient G1 cells.Since KEAP1 is involved in protein ubiquitylation, Orthwein et al. hypothesized that ubiquitylation of PALB2 in the BRCA1-interacting region might block their interaction. Indeed, mutation of lysines in the interacting region of PALB2 restored its interaction with BRCA1 in G1 cells. Furthermore, pull-down experiments showed that ubiquitylation of PALB2 on Lysine-20 by KEAP1-CRL3 prevented its interaction with BRCA1. However, as neither the activity of the KEAP1-CRL3 ubiquitin ligase nor its interaction with BRCA1 is cell cycle regulated, Orthwein et al. reasoned that a deubiquitylation step could be the rate-limiting regulator of the BRCA1-PALB2 interaction. They highlighted the deubiquitylating enzyme USP11 as a potential candidate for this activity due to its interaction with BRCA1, BRCA2 and PALB2, and indeed found that USP11 disruption impaired the interaction between BRCA1 and PALB2. Moreover, they found that USP11 was unstable and interacted poorly with PALB2 in G1 cells, and that USP11 was rapidly lost by proteasomal degradation in G1 phase after DNA damage. By contrast, expression of USP11 in S-phase was high and insensitive to DNA damage. Taken together, these data led the authors to propose that the opposing activities of USP11 and KEAP1-CRL3 regulate cell cycle-dependent interactions between BRCA1 and PALB2 (Figure 1).Open in a separate windowFigure 1Schematic representation indicating how the opposing activities of USP11 and KEAP1-CRL3 regulate cell cycle-dependent interactions between BRCA1 and PALB2, and thereby mediate pathway choice in DSB repair.To extend these remarkable observations, Orthwein et al. disrupted this regulatory network to allow HR in G1 cells. They expected that depletion of KEAP1 in 53BP1-deficient cells might be sufficient for RAD51 foci formation following ionizing radiation (IR), but this was not the case because end resection remained a limiting factor. To counteract this, the authors expressed a constitutively active form of CtIP (T847E)⁹, which augmented resection and led to the efficient formation of IR-induced RAD51 foci in 53BP1- and KEAP1-deficient G1 cells. To address whether these RAD51 foci in G1 cells corresponded to productive HR events, they used a fluorescent-based gene-targeting assay. Whereas CtIP (T847E)expressed in 53BP1-deficient cells alone was insufficient to induce productive HR, depletion of KEAP1 or expression of a non-ubiquitylable version of PALB2 led to a robust increase in gene-targeting events. Collectively, this study therefore demonstrates that activation of DNA end resection, combined with the recruitment of BRCA2 to DSBs, are both necessary and sufficient to produce HR in G1 cells.Gene targeting has great potential for therapeutic purposes, but the fact that most cells in the body are non-dividing has so far limited its use¹⁰. We suspect that the new knowledge highlighted in this work will further improve gene-targeting therapies to help fight human diseases.     

Designed for deconstruction--poplar trees altered in cell wall lignification improve the efficacy of bioethanol production

? There is a pressing global need to reduce the increasing societal reliance on petroleum and to develop a bio-based economy. At the forefront is the need to establish a sustainable, renewable, alternative energy sector. This includes liquid transportation fuel derived from lignocellulosic plant materials. However, one of the current limiting factors restricting the effective and efficient conversion of lignocellulosic residues is the recalcitrance of the substrate to enzymatic conversion. ? In an attempt to assess the impact of cell wall lignin on recalcitrance, we subjected poplar trees engineered with altered lignin content and composition to two potential industrial pretreatment regimes, and evaluated the overall efficacy of the bioconversion to ethanol process. ? It was apparent that total lignin content has a greater impact than monomer ratio (syringyl : guaiacyl) on both pretreatments. More importantly, low lignin plants showed as much as a 15% improvement in the efficiency of conversion, with near complete hydrolysis of the cellulosic polymer. ? Using genomic tools to breed or select for modifications in key cell wall chemical and/or ultrastructural traits can have a profound effect on bioenergy processing. These techniques may therefore offer means to overcome the current obstacles that underpin the recalcitrance of lignocellulosic substrates to bioconversion.     

Control of lactate production by Selenomonas ruminantium: homotropic activation of lactate dehydrogenase by pyruvate.

Selenomonas ruminantium produced one mole of D(-)-lactate per mole of glucose used at all dilution rates in ammonia-limited continuous culture. In contrast, lactate production varied according to the dilution rate when glucose was the limiting nutrient. At dilution rates of less than 0.2 h-1, acetate and propionate were the main fermentation products and lactate production was low. At dilution rates above 0.2 h-1, the pattern changed to one of high lactate production similar to that under ammonia limitation. Experiments with cell-free extracts of S. ruminantium showed that D(-)-lactate dehydrogenase had sigmoidal kinetics consistent with homotropic activation of the enzyme by its substrate, pyruvate. This feature allows S. ruminantium to amplify the effects of relatively small changes in the intracellular concentration of pyruvate to cause much larger changes in the rate of production of lactate. Some confirmation that this mechanism of control occurs under physiological conditions was obtained in glucose-limited culture, in which the sigmoidal increase in lactate production was accompanied by a linear increase in pyruvate excretion as the dilution rate increased.     

Immunoglobulin D switching can occur through homologous recombination in human B cells.

Prototypical class switching in mouse and human immunoglobulin heavy chains occurs through recombination of tandem blocks of short repeats located 5' to each heavy chain constant region (CH) except C delta. Deletion of C mu in immunoglobulin D (IgD)-secreting murine plasmacytomas occurs illegitimately. We demonstrate here that in human IgD-secreting myeloma cells freshly isolated from patient bone marrow and in normal peripheral blood B lymphocytes, an IgD switch can occur through homologous recombination of a direct repeat consisting of a 442-bp sequence 1.5 kbp 3' of the JH complex and a 443-bp sequence that is duplicated almost perfectly (96% similarity) 1.7 kbp 5' of the C delta gene (442/443-base-pair [bp] repeat). This homologous recombination mechanism is not exclusive for IgD switching, since C mu deletion endpoints in two established IgD-secreting myeloma cell lines fall outside the 442/443-bp repeat. The 442/443-bp mediated recombination shows cell type specificity, and we propose that it represents a unique mode for increased levels of IgD secretion in humans.