Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

BackgroundCultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by the Δ12 fatty acid desaturase (FAD) encoded by AhFAD2A and AhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C → A:T) of AhFAD2A and an “A” insertion of AhFAD2B resulted in high-oleic acid phenotype. Detection of AhFAD2 mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detect AhFAD2 genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detection AhFAD2 genotype of large number of breeding materials.ResultsHere, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC₄F₂ seeds was aabb.ConclusionsDue to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable for determining AhFAD2 genotype than other methods.     

Molecular characterization and genetic diversity of different genotypes of Oryza sativa and Oryza glaberrima

BackgroundAvailability of related rice species is critical for rice breeding and improvement. Two distinct species of domesticated rice exist in the genus Oryza: Oryza sativa (Asian rice) and Oryza glaberrima (African rice). New rice for Africa (NERICA) is derived from interspecific crosses between these two species. Molecular profiling of these germplasms is important for both genetics and breeding studies. We used 30 polymorphic SSR markers to assess the genetic diversity and molecular fingerprints of 53 rice genotypes of O. sativa, O. glaberrima, and NERICA.ResultsIn total, 180 alleles were detected. Average polymorphism information content and Shannon's information index were 0.638 and 1.390, respectively. Population structure and neighbor-joining phylogenetic tree revealed that 53 genotypes grouped into three distinct subpopulations conforming to the original three groups, except three varieties (IR66417, WAB450-4, MZCD74), and that NERICA showed a smaller genetic distance from O. sativa genotypes (0.774) than from O. glaberrima genotypes (0.889). A molecular fingerprint map of the 53 accessions was constructed with a novel encoding method based on the SSR polymorphic alleles. Ten specific SSR markers displayed different allelic profiles between the O. glaberrima and O. sativa genotypes.ConclusionsGenetic diversity studies revealed that 50 rice types were clustered into different subpopulations whereas three genotypes were admixtures. Molecular fingerprinting and 10 specific markers were obtained to identify the 53 rice genotypes. These results can facilitate the potential utilization of sibling species in rice breeding and molecular classification of O. sativa and O. glaberrima germplasms.     

Development and application of two novel functional molecular markers of BADH2 in rice

BackgroundFragrance is one of the most important quality traits in rice, and the phenotype is attributed to the loss-of-function betaine aldehyde dehydrogenase (BADH2) gene. At least 12 allelic variations of BADH2 have been identified, and some of these have been applied to rice fragrance breeding using traditional molecular markers and Sanger sequencing techniques. However, these traditional methods have several limitations, such as being very expensive, imprecise, inefficient, and having security issues. Thus, a new molecular marker technology must be developed to improve rice fragrance breeding.ResultsIn this study, more than 95% of the cultivated fragrant rice varieties belonged to a 7-bp deletion in exon 2 (badh2-E2) or an 8-bp deletion and 3-bp variation in exon 7 (badh2-E7). Both allelic variations resulted in the loss of function of the badh2 gene. We developed two novel SNP molecular markers, SNP_badh2-E2 and SNP_badh2-E7, related to the alleles. Their genotype and phenotype were highly cosegregated in the natural variation of rice accessions, with 160 of the 164 fragrant rice varieties detected with the two markers. These markers cosegregated with the fragrance phenotype in the F₂ population.ConclusionsTwo functional SNP molecular markers of badh2-E2 and badh2-E7 allelic variations were developed. These functional SNP molecular markers can be used for genotype and genetic improvement of rice fragrance through marker-assisted selection and will significantly improve the efficiency of fragrant rice breeding and promote commercial molecular breeding of rice in the future.How to cite: Li W, Zeng X, Li S, et al. Development and application of two novel functional molecular markers of BADH2 in rice. Electron J Biotechnol 2020;46. https://doi.org/10.1016/j.ejbt.2020.04.004.     

Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani

BackgroundRhizoctonia solani (teleomorph: Thanatephorus cucumeris) is one of the most important pathogens of rice (Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani, five methods, including E.Z.N.A.™ Fungal RNA Kit, sodium dodecyl sulfate (SDS)–sodium borate, SDS–polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study.ResultsThe electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS–sodium borate, SDS–PVP and E.Z.N.A.™ Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment.ConclusionIt is concluded that SDS–sodium borate and SDS–PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A.™ Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield.     

RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

BackgroundZymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments.ResultsThe plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4.ConclusionThis approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.     

Segregation distortion in homozygous lines obtained via anther culture and maize doubled haploid methods in comparison to single seed descent in wheat (Triticum aestivum L.)

BackgroundThe quality of wheat grain depends on several characteristics, among which the composition of high molecular weight glutenin subunits, encoded by Glu-1 loci, are the most important. Application of biotechnological tools to accelerate the attainment of homozygous lines may influence the proportion of segregated genotypes. The objective was to determine, whether the selection pressure generated by the methods based on in vitro cultures, may cause a loss of genotypes with desirable Glu-1 alleles.ResultsHomozygous lines were derived from six winter wheat crosses by pollination with maize (DH-MP), anther culture (DH-AC) and single seed descent (SSD) technique. Androgenetically-derived plants that originated from the same callus were examined before chromosome doubling using allele-specific and microsatellite markers. It was found that segregation distortion in SSD and DH-MP populations occurred only in one case, whereas in anther-derived lines they were observed in five out of six analyzed combinations.ConclusionsSegregation distortion in DH-AC populations was caused by the development of more than one plant of the same genotype from one callus. This distortion was minimized if only one plant per callus was included in the population. Selection of haploid wheat plants before chromosome doubling based on allele-specific markers allows us to choose genotypes that possess desirable Glu-1 alleles and to reduce the number of plants in the next steps of DH production. The SSD technique appeared to be the most advantageous in terms of Mendelian segregation, thus the occurrence of residual heterozygosity can be minimized by continuous selfing beyond the F₆ generation.     

Production of wheat-doubled haploids resistant to eyespot supported by marker-assisted selection

BackgroundWheat is one of the most important crops cultivated all over the world. New high-yielding cultivars that are more resistant to fungal diseases have been permanently developed. The present study aimed at the possibility of accelerating the process of breeding new cultivars, resistant to eyespot, by using doubled haploids (DH) system supported by marker-assisted selection.ResultsTwo highly resistant breeding lines (KBP 0916 and KBH 4942/05) carrying Pch1 gene were crossed with the elite wheat genotypes. Hybrid plants of early generations were analyzed using endopeptidase EpD1 and two SSR markers linked to the Pch1 locus. Selected homozygous and heterozygous genotypes for the Pch1-linked EpD1b allele were used to produce haploid plants. Molecular analyses were performed on haploids to identify plants possessing Pch1 gene. Chromosome doubling was performed only on haploid plants with Pch1 gene. Finally, 65 DH lines carrying eyespot resistance gene Pch1 and 30 lines without this gene were chosen for the eyespot resistance phenotyping in a field experiment.ConclusionsResults of the experiment confirmed higher resistance to eyespot of the genotypes with Pch1 in comparison to those without this gene. This indicates the efficiency of selection at the haploid level.How to cite: Wiśniewska H, Majka M, Kwiatek M, et al. Production of wheat doubled haploids resistant to eyespot supported by marker-assisted selection. Electron J Biotechnol 2019;37. https://doi.org/10.1016/j.ejbt.2018.10.003     

Identification and characterization of profilin gene family in rice

BackgroundProfilin proteins (PRFs) are small (12–15 kD) actin-binding protein, which play a significant role in cytoskeleton dynamics and plant development via regulating actin polymerization. Profilins have been well documented in Arabidopsis, Zea mays L. as well as Phaseolus vulgaris, however no such fully characterization of rice (Oryza sativa L.) profilin gene family has been reported thus far.ResultIn the present study, a comprehensive genome-wide analysis of rice PRF genes was completed and three members were identified. OsPRF1 and OsPRF2 shared 98.5% similarity (6 nucleotide divergence), but the deduced amino acid sequences of OsPRF1 and OsPRF2 are fully identical. In contrast, the OsPRF3 presents relatively lower similarity with OsPRF1 and OsPRF2. Phylogenetic analysis also support that OsPRF1 has a closer relationship with OsPRF2. Expression pattern analysis revealed the differential expression of OsPRFs in tissues of mature plant, which suggested the potential spatial functional specificity for rice profilin genes. Subcellular localization analysis revealed the OsPRFs were localized in cytoplasm and nucleus and all of them could bind actin monomers. Furthermore, abiotic stresses and hormones treatments assay indicated that the three OsPRF genes could be differentially regulated, suggesting that OsPRF genes might participate in different stress processes in rice.ConclusionsTaken together, our study provides a comprehensive analysis of the OsPRF gene family and will provide a basis for further studies on their roles in rice development and in response to abiotic stresses.How to cite: Zhang Y, Dong G, Wu L, et al. Identification and characterization of profilin gene family in rice. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.08.004.     

Analysis of gene expression profiles in response to Sporisorium reilianum f. sp. zeae in maize (Zea mays L.)

BackgroundHead smut of maize, which is caused by Sporisorium reilianum f. sp. zeae (Kühn), is a serious disease in maize. In order to reveal the molecular mechanism of the resistance to head smut in maize, a microarray containing ∼ 14,850 probes was used to monitor the gene expression profiles between a disease resistant near isogenic line (NIL) and a highly susceptible inbred line after S. reilianum was injected with an artificial inoculation method.ResultsLevels of expression for 3,532 genes accounting for 23.8% of the total probes changed after inoculation. Gene Ontology analysis revealed that the differentially expressed genes participated in physiological and biochemical pathways. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that plant–pathogen interaction, natural killer cell mediated cytotoxicity and benzoxazinoid biosynthesis pathways play important roles in resistance to head smut. Three head smut resistance-related candidate genes, CLAVATA1, bassinosteroid insensitive 1-associated receptor kinase 1 and LOC100217307 with leucine-rich repeat (LRR) conserved domains were identified, each of which is in maize mapping bin 2.09, a region previously shown to include a major QTL for head smut resistance. Furthermore, LOC100217307 was validated by quantitative real-time (qRT)-PCR inferring that this gene may be involved in the resistance to head smut of maize.ConclusionsThis study provided valuable information for cloning, functional analysis and marker assisted breeding of head smut resistance genes.     

Prevalence of K-ras Codon 12 Mutations in Indian Patients with Head and Neck Cancer

In human tumors, somatic mutation frequently occur in K-ras gene at codon 12, which makes the K-ras protein hyper active leading to uncontrolled signaling for cell division: one of the important hall mark of cancer. In order to correlate mutations in K-ras to cause, response to treatment, disease progression and recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) the following study was undertaken. By using PCR–RFLP method prevalence of codon 12 in K-ras gene was studied in 56 HNSCC patients. High frequency of K-ras mutation was detected in codon 12 (60.71%). The result of this study helps us in understanding the role of K-ras somatic mutations in HNSCC patients and in designing novel treatment protocols for HNSCC patients.Electronic supplementary materialThe online version of this article (10.1007/s12291-020-00882-w) contains supplementary material, which is available to authorized users.     

Characterization and properties of the biosurfactant produced by Candida lipolytica UCP 0988

BackgroundBiotechnological processes are costly, especially for the production of biosurfactants. The successful production of a biosurfactant is dependent on the development of processes using low cost raw materials. Considering the importance of the characteristics of a biosurfactant to facilitate its industrial application, the properties of the biosurfactant produced by Candida lipolytica through previously optimized medium have been established.ResultsThe yeast was grown for 72 h to determine the kinetics of growth and production. The surface tension of the cell-free broth was reduced from 55 to 25 mN/m. The yield of biosurfactant was 8.0 g/l with a CMC of 0.03%. The biosurfactant was characterized as an anionic lipopeptide composed of 50% protein, 20% lipids, and 8% of carbohydrates.ConclusionsThe isolated biosurfactant showed no toxicity against different vegetable seeds: Brassica oleracea, Solanum gilo and Lactuca sativa L. and the micro-crustacean Artemia salina. The properties of the biosurfactant produced suggest its potential application in industries that require the use of effective compounds at low cost.     

Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

BackgroundIn industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze–thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain.ResultsElite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze–thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield.ConclusionsA multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze–thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.How to cite: Zhang Q, Jin Y, Fang Y, et al. Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high gravity bioethanol fermentation. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.06.003     

Data mining and influential analysis of gene expression data for plant resistance gene identification in tomato (Solanum lycopersicum)

BackgroundMolecular mechanisms of plant–pathogen interactions have been studied thoroughly but much about them is still unknown. A better understanding of these mechanisms and the detection of new resistance genes can improve crop production and food supply. Extracting this knowledge from available genomic data is a challenging task.ResultsHere, we evaluate the usefulness of clustering, data-mining and regression to identify potential new resistance genes. Three types of analyses were conducted separately over two conditions, tomatoes inoculated with Phytophthora infestans and not inoculated tomatoes. Predictions for 10 new resistance genes obtained by all applied methods were selected as being the most reliable and are therefore reported as potential resistance genes.ConclusionApplication of different statistical analyses to detect potential resistance genes reliably has shown to conduct interesting results that improve knowledge on molecular mechanisms of plant resistance to pathogens.     

Development of SSR marker by RNA-seq and its application in genotyping pearl sac in pearl oyster Pinctada fucata martensii

BackgroundPearl oyster Pinctada fucata martensii is cultured for producing round nucleated pearls. Pearl production involves a surgical operation where a mantle tissue graft from a donor oyster and a round nucleus are implanted in the gonad of a host oyster. Whether the mantle graft implanted in the gonad of a host oyster contributes to the formation of a pearl sac that secretes pearl nacre to form a pearl should be determined. In April 2012, two full-sib families were separately used as donor and host oysters for a nucleus insertion operation. The pearl sac was sampled from the host oysters at day 60 after nucleus operation. A large number of simple sequence repeat (SSR) markers were developed using Illumina HiSeq™ 2000 platform. The two full-sib families were also used to mine diagnostic SSR markers for genotyping donor oyster, host oyster, and pearl sac.ResultsA total of 3168 microsatellite loci were identified in 39,078 unigenes, and 1977 SSR primers were designed by Primer 3.0. Forty-seven SSR primers were validated, and the rate of successful amplification was 72.3%. Two diagnostic SSR primers could successfully genotype pearl sac, donor oyster, and host oyster. Donor and host oysters were both homogenous, and the alleles in pearl sac were identical to those in donor and host oysters.ConclusionsThe present results confirmed that the mantle graft implanted in the gonad of host oyster contributed to the formation of the pearl sac in pearl oyster P. fucata martensii.     

Dynamic changes of total acid and bacterial communities during the traditional fermentation of Hong Qu glutinous rice wine

BackgroundHong Qu glutinous rice wine (HQGRW) is brewed under non-aseptic fermentation conditions, so it usually has a relatively high total acid content. The aim of this study was to investigate the dynamics of the bacterial communities and total acid during the fermentation of HQGRW and elucidate the correlation between total acid and bacterial communities.ResultsThe results showed that the period of rapid acid increase during fermentation occurred at the early stage of fermentation. There was a negative response between total acid increase and the rate of increase in alcohol during the early fermentation stage. Bacterial community analysis using high-throughput sequencing technology was found that the dominant bacterial communities changed during the traditional fermentation of HQGRW. Both principal component analysis (PCA) and hierarchical clustering analysis revealed that there was a great difference between the bacterial communities of Hong Qu starter and those identified during the fermentation process. Furthermore, the key bacteria likely to be associated with total acid were identified by Spearman's correlation analysis. Lactobacillus, unclassified Lactobacillaceae, and Pediococcus were found, which can make significant contributions to the total acid development (| r | > 0.6 with FDR adjusted P < 0.05), establishing that these bacteria can associate closely with the total acid of rice wine.ConclusionsThis was the first study to investigate the correlation between bacterial communities and total acid during the fermentation of HQGRW. These findings may be helpful in the development of a set of fermentation techniques for controlling total acid.How to cite: Liang Z, Lin X, He Z, et al. Dynamic changes of total acid and bacterial communities during the traditional fermentation of Hong Qu glutinous rice wine. Electron J Biotechnol 2020;43. https://doi.org/10.1016/j.ejbt.2019.12.002     

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid,Myzus persicae

BackgroundPiercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plant-derived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns.ResultsPinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP-9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids.ConclusionsThese findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloem-specific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.How to citeUmer N, Naqvi RZ, Rauf I, et al. Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.07.004.     

Statistical optimization of cellulases by Talaromyces thermophilus utilizing Saccharum spontaneum,a novel substrate

BackgroundAt present, cellulases are the most important enzymes worldwide, and their demand has been increasing in the industrial sector owing to their notable hydrolysis capability.ResultsIn the present study, contrary to conventional techniques, three physical parameters were statistically optimized for the production of cellulase by thermophilic fungi by using response surface methodology (RSM). Among all the tested thermophilic strains, the best cellulase producing fungus was identified as Talaromyces thermophilus – both morphologically and molecularly through 5.8S/ITS rDNA sequencing. The central composite design (CCD) was used to evaluate the interactive effect of the significant factors. The CCD was applied by considering incubation period, pH, and temperature as the model factors for the present investigation. A second-order quadratic model and response surface method revealed that the independent variables including pH 6, temperature 50 °C, and incubation period 72 h significantly influenced the production of cellulases. The analysis of variance (ANOVA) indicated that the established model was significant (P ≤ 0.05) and showed the high adequacy of the model. The actual and predicted values of CMCase and FPase activity showed good agreement with each other and also confirmed the validity of the designed model.ConclusionsWe believe the present findings to be the first report on cellulase production by exploiting Kans grass (Saccharum spontaneum) as a substrate through response surface methodology by using thermophilic fungus, Talaromyces thermophilus.How to cite: Abdullah R, Tahseen M, Nisar K et al. Statistical optimization of cellulases by Talaromyces thermophilus utilizing Saccharum spontaneum, a novel substrate. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.007.     

Biosynthetic L-tert-leucine using Escherichia coli co-expressing a novel NADH-dependent leucine dehydrogenase and a formate dehydrogenase

BackgroundL-tert-Leucine has been widely used in pharmaceutical, chemical, and other industries as a vital chiral intermediate. Compared with chemical methods, enzymatic methods to produce L-tert-leucine have unparalleled advantages. Previously, we found a novel leucine dehydrogenase from the halophilic thermophile Laceyella sacchari (LsLeuDH) that showed good thermostability and great potential for the synthesis of L-tert-leucine in the preliminary study. Hence, we manage to use the LsLeuDH coupling with a formate dehydrogenase from Candida boidinii (CbFDH) in the biosynthesis of L-tert-leucine through reductive amination in the present study.ResultThe double-plasmid recombinant strain exhibited higher conversion than the single-plasmid recombinant strain when resting cells cultivated in shake flask for 22 h were used. Under the optimized conditions, the double-plasmid recombinant E. coli BL21 (pETDute-FDH-LDH, pACYCDute-FDH) transformed 1 mol·L^-1 trimethylpyruvate (TMP) completely into L-tert-leucine with greater than 99.9% ee within 8 h.ConclusionsThe LsLeuDH showed great ability to biosynthesize L-tert-leucine. In addition, it provided a new option for the biosynthesis of L-tert-leucine.How to citeWang L, Zhu W, Gao Z, et al. Biosynthetic L-tert-leucine using Escherichia coli co-expressing a novel NADH-dependent leucine dehydrogenase and a formate dehydrogenase. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.07.001