Native Alleles Research Articles

Integrating morphological, molecular and cytogenetic data for F2 sea turtle hybrids diagnosis revealed balanced chromosomal sets.

Hybridization could be considered part of the evolutionary history of many species. The hybridization among sea turtle species on the Brazilian coast is atypical and occurs where nesting areas and reproductive seasons overlap. Integrated analysis of morphology and genetics is still scarce, and there is no evidence of the parental chromosome set distribution in sea turtle interspecific hybrids. In this study, chromosome markers previously established for pure sea turtle species were combined with morphological and molecular analyses aiming to recognize genetic composition and chromosome sets in possible interspecific hybrids initially identified by mixed morphology. The data showed that one hybrid could be an F2 individual among Caretta caretta × Eretmochelys imbricata × Chelonia mydas, and another is resulting from backcross between C. caretta × Lepidochelys olivacea. Native alleles of different parental lineages were reported in the hybrids, and, despite this, it was verified that the hybrid chromosome sets were still balanced. Thus, how sea turtle hybridism can affect genetic features in the long term is a concern, as the implications of the crossing-over in hybrid chromosomal sets and the effects on genetic function are still unpredictable.

A polygenic architecture with habitat-dependent effects underlies ecological differentiation in Silene.

Summary Ecological differentiation can drive speciation but it is unclear how the genetic architecture of habitat‐dependent fitness contributes to lineage divergence. We investigated the genetic architecture of cumulative flowering, a fitness component, in second‐generation hybrids between Silene dioica and Silene latifolia transplanted into the natural habitat of each species.We used reduced‐representation sequencing and Bayesian sparse linear mixed models (BSLMMs) to analyze the genetic control of cumulative flowering in each habitat.Our results point to a polygenic architecture of cumulative flowering. Allelic effects were mostly beneficial or deleterious in one habitat and neutral in the other. Positive‐effect alleles often were derived from the native species, whereas negative‐effect alleles, at other loci, tended to originate from the non‐native species.We conclude that ecological differentiation is governed and maintained by many loci with small, habitat‐dependent effects consistent with conditional neutrality. This pattern may result from differences in selection targets in the two habitats and from environmentally dependent deleterious load. Our results further suggest that selection for native alleles and against non‐native alleles acts as a barrier to gene flow between species.

Locus-specific introgression in young hybrid swarms: Drift may dominate selection.

Closely related species that have previously inhabited geographically separated ranges are hybridizing at an increasing rate due to human disruptions. These human-mediated hybrid zones can be used to study reproductive isolation between species at secondary contact, including examining locus-specific rates of introgression. Introgression is expected to be heterogenous across the genome, reflecting variation in selection. Those loci that introgress especially slowly are good candidates for being involved in reproductive isolation, while those loci that introgress quickly may be involved in adaptive introgression. In the context of conservation, policy makers are especially concerned about introduced alleles moving quickly into the background of a native or endemic species, as these alleles could replace the native alleles in the population, leading to extinction via hybridization. We applied genomic cline analyses to 44,997 SNPs to identify loci introgressing more or less when compared to the genome wide expectation in a human-mediated hybridizing population of red deer and sika in Kintyre Scotland. We found 11.4% of SNPs had cline centres that were significantly different from the genome wide expectation, and 17.6% of all SNPs had excess rates of introgression. Based on simulations, we believe that many of these markers have diverged from the genome-wide average due to drift, rather than because of selection, and we suggest that these simulations can be useful as a null distribution for future studies of genomic clines. Future work on red deer and sika could determine the policy implications of allelic-replacement due to drift rather than selection, and could use replicate, geographically distinct hybrid zones to narrow down those loci that are responding to selection.

Biased Gene Retention in the Face of Introgression Obscures Species Relationships.

Phylogenomic analyses are recovering previously hidden histories of hybridization, revealing the genomic consequences of these events on the architecture of extant genomes. We applied phylogenomic techniques and several complementary statistical tests to show that introgressive hybridization appears to have occurred between close relatives of Arabidopsis, resulting in cytonuclear discordance and impacting our understanding of species relationships in the group. The composition of introgressed and retained genes indicates that selection against incompatible cytonuclear and nuclear–nuclear interactions likely acted during introgression, whereas linkage also contributed to genome composition through the retention of ancient haplotype blocks. We also applied divergence-based tests to determine the species branching order and distinguish donor from recipient lineages. Surprisingly, these analyses suggest that cytonuclear discordance arose via extensive nuclear, rather than cytoplasmic, introgression. If true, this would mean that most of the nuclear genome was displaced during introgression whereas only a small proportion of native alleles were retained.

Maize BIG GRAIN1 homolog overexpression increases maize grain yield.

SummaryThe Zea Mays BIG GRAIN 1 HOMOLOG 1 (ZM‐BG1H1) was ectopically expressed in maize. Elite commercial hybrid germplasm was yield tested in diverse field environment locations representing commercial models. Yield was measured in 101 tests across all 4 events, 26 locations over 2 years, for an average yield gain of 355 kg/ha (5.65 bu/ac) above control, with 83% tests broadly showing yield gains (range +2272 kg/ha to −1240 kg/ha), with seven tests gaining more than one metric ton per hectare. Plant and ear height were slightly elevated, and ear and tassel flowering time were delayed one day, but ASI was unchanged, and these traits did not correlate to yield gain. ZM‐BG1H1 overexpression is associated with increased ear kernel row number and total ear kernel number and mass, but individual kernels trended slightly smaller and less dense. The ZM‐BG1H1 protein is detected in the plasma membrane like rice OS‐BG1. Five predominant native ZM‐BG1H1 alleles exhibit little structural and expression variation compared to the large increased expression conferred by these ectopic alleles.

Implementation of advanced Optimum Contribution Selection in small-scale breeding schemes: prospects and challenges in Vorderwald cattle

Vorderwald cattle are a regional cattle breed from the Black Forest in south western Germany. In recent decades, commercial breeds have been introgressed to upgrade the breed in performance traits. On one hand, native genetic diversity of the breed should be conserved. On the other hand, moderate rates of genetic gain are needed to satisfy breeders to keep the breed. These goals are antagonistic, since the native proportion of the gene pool is negatively correlated to performance traits and the carriers of introgressed alleles are less related to the population. Thus, a standard Optimum Contribution Selection (OCS) approach would lead to reinforced selection on migrant contributions (MC). Our objective was the development of strategies for practical implementation of an OCS approach to manage the MC and native genetic diversity of regional breeds. Additionally, we examined the organisational efforts and the financial impacts on the breeding scheme of Vorderwald cattle. We chose the advanced Optimum Contribution Selection (aOCS) to manage the breed in stochastic simulations based on real pedigree data. In addition to standard OCS approaches, aOCS facilitates the management of the MC and the rate of inbreeding at native alleles. We examined two aOCS strategies. Both strategies maximised genetic gain, while strategy (I) conserved the MC in the breeding population and strategy (II) reduced the MC at a predefined annual rate. These two approaches were combined with one of three flows of replacement of sires (FoR strategies). Additionally, we compared breeding costs to clarify about the financial impact of implementing aOCS in a young sire breeding scheme. According to our results, conserving the MC in the population led to significantly (P < 0.01) higher genetic gain (1.16 ± 0.13 points/year) than reducing the MC (0.88 ± 0.10 points/year). In simulation scenarios that conserved the MC, the final value of MC was 57.6% ± 0.004, while being constraint to 58.2%. However, reducing the MC is only partially feasible based on pedigree data. Additionally, this study proves that the classical rate of inbreeding can be managed by constraining only the rate of inbreeding at native alleles within the aOCS approach. The financial comparison of the different breeding schemes proved the feasibility of implementing aOCS in Vorderwald cattle. Implementing the modelled breeding scheme would reduce costs by 1.1% compared with the actual scheme. Reduced costs were underpinned by additional genetic gain in superior simulation scenarios compared to expected genetic gain in reality (+4.85%).

Optimum contribution selection for animal breeding and conservation: the R package optiSel

BackgroundSelecting animals for breeding in the optimum way plays an essential role for the management of genetic resources and in selective breeding of livestock species. It requires to compute the optimum genetic contribution of each selection candidate to the next generation. Current software packages for optimum contribution selection (OCS) are not able to handle the main conflicting objectives of animal breeding programs simultaneously, which includes to increase genetic gain, to increase or to maintain genetic diversity, to recover the original genetic background of endangered breeds with historic introgression, and to maintain or increase genetic diversity at native alleles.ResultsThe free R package optiSel offers functions for estimating the above mentioned parameters from pedigree and marker data, and for solving OCS problems. One parameter can be optimized, whereas the remaining ones can be constrained. The results reveal the optimum numbers of offspring of all selection candidates, and can subsequently be used for mate allocation. Different solvers can be used. Solver slsqp was superior when the genetic diversity at native alleles was to be maximized, whereas solvers cccp and cccp2 were superior for all other OCS problems.ConclusionOptimum contribution selection applied to local breeds requires special attention due to the conflicting objectives of their breeding programs. The free R package optiSel is an easy-to-use software taking these conflicting objectives into account.

Hybridization, coexistence, and possible reproductive interference between native Oxalis corniculata and alien O. dillenii in Japan

Hybridization with alien species can affect the reproduction and persistence of native species. Native Oxalis corniculata and alien O. dillenii are distributed in human-made habitats in Japan, and they coexist with their putative hybrid. We examined the origin of the putative hybrid and the possibility of introgression and reproductive interference between these species by analyzing their morphological, cytological, genetic, and reproductive characteristics. The flower morphology of the putative hybrid was similar to the alien species. The chromosome complement and the amount of nuclear DNA of the putative hybrid were intermediate between the two species. Genotyping of the nuclear ITS region showed the putative hybrid was invariably heterozygous for native and alien alleles. Haplotyping of the chloroplast trnS–trnG region showed that 22 and four hybrids had their maternal origin in the alien and native species, respectively. The ovules of the putative hybrid were sterile. When pollen of the putative hybrid was applied to flowers of the parental species, the fruit set was very low, suggesting that their pollen is only marginally viable. Based on these results, we conclude that the putative hybrid is an F1 hybrid between native and alien Oxalis species. The hybrid is virtually sterile, indicating that hybridization led to a waste of gametes and may result in reproductive interference between the two parental species. The capacity for selfing and/or asexual reproduction may weaken reproductive interference between the species; alternatively, their apparent coexistence may be maintained by dispersal from different parental habitats.

Reconstruction of the Vancomycin-Susceptible Staphylococcus aureus Phenotype From a Vancomycin-Intermediate S. aureus XN108.

The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) has raised healthcare concerns worldwide. VISA is often associated with multiple genetic changes. However, the relative contributions of these changes to VISA phenotypes are incompletely defined. We have characterized VISA XN108 with vancomycin MIC of 12 μg/ml. Genome comparison revealed that WalK(S221P), GraS(T136I), and RpoB(H481N) mutations possibly contributed to the VISA phenotype of XN108. In this study, the above mutations were stepwise cured, and the phenotypes between XN108 and its derivates were compared. We constructed four isogenic mutant strains, XN108-WalK(P221S) (termed as K65), XN108-GraS(I136T) (termed as S65), XN108-RpoB(N481H) (termed as B65), and XN108-WalK(P221S)/GraS(I136T) (termed as KS65), using the allelic replacement experiments with the native alleles derived from a vancomycin-susceptible S. aureus isolate DP65. Antimicrobial susceptibility test revealed K65 and S65 exhibited decreased vancomycin resistance, whereas B65 revealed negligibly differed when compared with the wild-type XN108. Sequentially introducing WalK(P221S) and GraS(I136T) completely converted XN108 into a VSSA phenotype. Transmission electronic microscopy and autolysis determination demonstrated that cell wall thickening and decreasing autolysis were associated with the change of vancomycin resistance levels. Compared with XN108, K65 exhibited 577 differentially expressed genes (DEGs), whereas KS65 presented 555 DEGs. Of those DEGs, 390 were common in K65 and KS65, including those upregulated genes responsible for citrate cycle and bacterial autolysis, and the downregulated genes involved in peptidoglycan biosynthesis and teichoic acid modification. In conclusion, a VSSA phenotype could be completely reconstituted from a VISA strain XN108. WalK(S221P) and GraS(T136I) mutations may work synergistically in conferring vancomycin resistance in XN108.

Autophagy gene overexpression in Saccharomyces cerevisiae perturbs subcellular organellar function and accumulates ROS to accelerate cell death with relevance to sparkling wine production

Traditional sparkling wines are produced by the refermentation of a base wine with yeast in the bottle followed by a critical period of aging. During the often lengthy aging process, yeast undergoes cell death and autolysis to release cellular compounds that over time ultimately contribute to the flavor and appearance of the product. While accelerating yeast autolysis for sparkling wine production has been the focus of several studies, employing overexpressed native yeast alleles for this purpose remains poorly explored. Here, we show that the overexpression of native yeast genes, specifically selected autophagic genes, results in accelerated cell death in nitrogen starvation and base wine refermentation. We show ATG3 or ATG4 overexpression has pleiotropic intracellular ramifications including reduced turnover of autophagic cargo, vacuolar fragmentation, abnormal accumulation of lipids, and accelerated accumulation of reactive oxygen species (ROS), all of which precede accelerated cell death. Our findings suggest that the increased expression of autophagy-related genes, such as ATG3 and ATG4, in industrial wine yeast can serve as a suitable marker or breeding strategy to accelerate the cell death and autolysis of wine yeast during sparkling wine production.

Alteration of TAC1 expression in Prunus species leads to pleiotropic shoot phenotypes

Prunus persica (peach) trees carrying the “Pillar” or “Broomy” trait (br) have vertically oriented branches caused by loss-of-function mutations in a gene called TILLER ANGLE CONTROL 1 (TAC1). TAC1 encodes a protein in the IGT gene family that includes LAZY1 and DEEPER ROOTING 1 (DRO1), which regulate lateral branch and root orientations, respectively. Here we found that some of the native TAC1 alleles in the hexaploid plum species Prunus domestica, which has a naturally more upright stature, contained a variable length trinucleotide repeat within the same exon 3 region previously found to be disrupted in pillar peach trees. RNAi silencing of TAC1 in plum resulted in trees with severely vertical branch orientations similar to those in pillar peaches but with an even narrower profile. In contrast, PpeTAC1 overexpression in plum led to trees with wider branch angles and more horizontal branch orientations. Pillar peach trees and transgenic plum lines exhibited pleiotropic phenotypes, including differences in trunk and branch diameter, stem growth, and twisting branch phenotypes. Expression profiling of pillar peach trees revealed differential expression of numerous genes associated with biotic and abiotic stress, hormone responses, plastids, reactive oxygen, secondary, and cell wall metabolism. Collectively, the data provide important clues for understanding TAC1 function and show that alteration of TAC1 expression may have broad applicability to agricultural and ornamental tree industries.

Genetic diversity and historic introgression in German Angler and Red Dual Purpose cattle and possibilities to reverse introgression

ABSTRACTRecovering native uniqueness has major importance for breeds with historic introgression. The aim of the study was to estimate population genetic parameters for two local red cattle breeds from Northern Germany and to study possibilities to reverse introgression. Genealogical information consisted of 90,783 individuals for German Angler and 187,255 individuals for Red Dual-Purpose cattle breed, with additional information on sex, born, breed, status, and conventional breeding values. It is concluded that the native genetic contribution could be included as an additional trait in the total merit index in order to recover a part of the native genetic background. Native contributions should be estimated in the long term from marker data in order to account for Mendelian sampling. The maintenance of a sufficient genetic diversity of native alleles can be achieved by an advanced OCS with appropriate constraints.

Long-Term Impact of Optimum Contribution Selection Strategies on Local Livestock Breeds with Historical Introgression Using the Example of German Angler Cattle.

The long-term performance of different selection strategies was evaluated via simulation using the example of a local cattle breed, German Angler cattle. Different optimum contribution selection (OCS) approaches to maximize genetic gain were compared to a reference scenario without selection and truncation selection. The kinships and migrant contribution (MC) were estimated from genomic data. Truncation selection achieved the highest genetic gain but decreased diversity considerably at native alleles. It also caused the highest increase in MCs. Traditional OCS, which only constrains kinship, achieved almost the same genetic gain but also caused a small increase of MC and remarkably reduced the diversity of native alleles. When MC was required not to increase and the increase of kinship at native alleles was restricted, the MC levels and the diversity at native alleles were well managed, and the genetic gain was only slightly reduced. However, genetic progress was substantially lower in the scenario that aimed to recover the original genetic background. Truncation selection and traditional OCS selection both reduce the genetic originality of breeds with historical introgression. The inclusion of MC and kinship at native alleles as additional constraints in OCS showed great potential for conservation. Recovery of the original genetic background is possible but requires many generations of selection and reduces the genetic progress in performance traits. Hence, constraining MCs at their current values can be recommended to avoid further reduction of genetic originality.

Novel optimum contribution selection methods accounting for conflicting objectives in breeding programs for livestock breeds with historical migration

BackgroundOptimum contribution selection (OCS) is effective for increasing genetic gain, controlling the rate of inbreeding and enables maintenance of genetic diversity. However, this diversity may be caused by high migrant contributions (MC) in the population due to introgression of genetic material from other breeds, which can threaten the conservation of small local populations. Therefore, breeding objectives should not only focus on increasing genetic gains but also on maintaining genetic originality and diversity of native alleles. This study aimed at investigating whether OCS was improved by including MC and modified kinships that account for breed origin of alleles. Three objective functions were considered for minimizing kinship, minimizing MC and maximizing genetic gain in the offspring generation, and we investigated their effects on German Angler and Vorderwald cattle.ResultsIn most scenarios, the results were similar for Angler and Vorderwald cattle. A significant positive correlation between MC and estimated breeding values of the selection candidates was observed for both breeds, thus traditional OCS would increase MC. Optimization was performed under the condition that the rate of inbreeding did not exceed 1% and at least 30% of the maximum progress was achieved for all other criteria. Although traditional OCS provided the highest breeding values under restriction of classical kinship, the magnitude of MC in the progeny generation was not controlled. When MC were constrained or minimized, the kinship at native alleles increased compared to the reference scenario. Thus, in addition to constraining MC, constraining kinship at native alleles is required to ensure that native genetic diversity is maintained. When kinship at native alleles was constrained, the classical kinship was automatically lowered in most cases and more sires were selected. However, the average breeding value in the next generation was also lower than that obtained with traditional OCS.ConclusionsFor local breeds with historical introgressions, current breeding programs should focus on increasing genetic gain and controlling inbreeding, as well as maintaining the genetic originality of the breeds and the diversity of native alleles via the inclusion of MC and kinship at native alleles in the OCS process.

Decreased platelet responsiveness to clopidogrel correlates with CYP2C19 and PON1 polymorphisms in atherosclerotic patients.

Clopidogrel and aspirin are the most commonly used medications worldwide for dual antiplatelet therapy after percutaneous coronary intervention. However, clopidogrel hyporesponsiveness related to gene polymorphisms is a concern. Populations with higher degrees of genetic admixture may have increased prevalence of clopidogrel hyporesponsiveness. To assess this, we genotyped CYP2C19, ABCB1, and PON1 in 187 patients who underwent percutaneous coronary intervention. Race was self-defined by patients. We also performed light transmission aggregometry with adenosine diphosphate (ADP) and arachidonic acid during dual antiplatelet therapy. We found a significant difference for presence of the CYP2C19*2 polymorphism between white and non-white patients. Although 7% of patients had platelet resistance to clopidogrel, this did not correlate with any of the tested genetic polymorphisms. We did not find platelet resistance to aspirin in this cohort. Multivariate analysis showed that patients with PON1 and CYP2C19 polymorphisms had higher light transmission after ADP aggregometry than patients with native alleles. There was no preponderance of any race in patients with higher light transmission aggregometry. In brief, PON1 and CYP2C19 polymorphisms were associated with lower clopidogrel responsiveness in this sample. Despite differences in CYP2C19 polymorphisms across white and non-white patients, genetic admixture by itself was not able to identify clopidogrel hyporesponsiveness.

Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles.

Asymmetric parental genome engineering by Cas9 during mouse meiotic exit.

Mammalian genomes can be edited by injecting pronuclear embryos with Cas9 cRNA and guide RNA (gRNA) but it is unknown whether editing can also occur during the onset of embryonic development, prior to pronuclear embryogenesis. We here report Cas9-mediated editing during sperm-induced meiotic exit and the initiation of development. Injection of unfertilized, mouse metaphase II (mII) oocytes with Cas9 cRNA, gRNA and sperm enabled efficient editing of transgenic and native alleles. Pre-loading oocytes with Cas9 increased sensitivity to gRNA ~100-fold. Paternal allelic editing occurred as an early event: single embryo genome analysis revealed editing within 3 h of sperm injection, coinciding with sperm chromatin decondensation during the gamete-to-embryo transition but prior to pronucleus formation. Maternal alleles underwent editing after the first round of DNA replication, resulting in mosaicism. Asymmetric editing of maternal and paternal alleles suggests a novel strategy for discriminatory targeting of parental genomes.

Immunogenicity of a synthetic vaccine based on Plasmodium vivax Duffy binding protein region II.

Molecules that play a role in Plasmodium merozoite invasion of host red blood cells represent attractive targets for blood-stage vaccine development against malaria. In Plasmodium vivax, merozoite invasion of reticulocytes is mediated by the Duffy binding protein (DBP), which interacts with its cognate receptor, the Duffy antigen receptor for chemokines, on the surface of reticulocytes. The DBP ligand domain, known as region II (DBPII), contains the critical residues for receptor recognition, making it a prime target for vaccine development against blood-stage vivax malaria. In natural infections, DBP is weakly immunogenic and DBPII allelic variation is associated with strain-specific immunity, which may compromise vaccine efficacy. In a previous study, a synthetic vaccine termed DEKnull that lacked an immunodominant variant epitope in DBPII induced functional antibodies to shared neutralizing epitopes on the native Sal1 allele. Anti-DEKnull antibody titers were lower than anti-Sal1 titers but produced more consistent, strain-transcending anti-DBPII inhibitory responses. In this study, we further characterized the immunogenicity of DEKnull, finding that immunization with recombinant DEKnull produced an immune response comparable to that obtained with native recombinant DBP alleles. Further investigation of DEKnull is necessary to enhance its immunogenicity and broaden its specificity.

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays AR GOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer's modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer's female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance.

Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of l-malic acid

Malic acid, a petroleum-derived C4-dicarboxylic acid that is used in the food and beverage industries, is also produced by a number of microorganisms that follow a variety of metabolic routes. Several members of the genus Aspergillus utilize a two-step cytosolic pathway from pyruvate to malate known as the reductive tricarboxylic acid (rTCA) pathway. This simple and efficient pathway has a maximum theoretical yield of 2 mol malate/mol glucose when the starting pyruvate originates from glycolysis. Production of malic acid by Aspergillus oryzae NRRL 3488 was first improved by overexpression of a native C4-dicarboxylate transporter, leading to a greater than twofold increase in the rate of malate production. Overexpression of the native cytosolic alleles of pyruvate carboxylase and malate dehydrogenase, comprising the rTCA pathway, in conjunction with the transporter resulted in an additional 27 % increase in malate production rate. A strain overexpressing all three genes achieved a malate titer of 154 g/L in 164 h, corresponding to a production rate of 0.94 g/L/h, with an associated yield on glucose of 1.38 mol/mol (69 % of the theoretical maximum). This rate of malate production is the highest reported for any microbial system.