Engineering co-utilization of glucose and xylose for chemical overproduction from lignocellulose.

Bio-refining lignocellulose could provide a sustainable supply of fuels and fine chemicals; however, the challenges associated with the co-utilization of xylose and glucose typically compromise the efficiency of lignocellulose conversion. Here we engineered the industrial yeast Ogataea polymorpha (Hansenula polymorpha) for lignocellulose biorefinery by facilitating the co-utilization of glucose and xylose to optimize the production of free fatty acids (FFAs) and 3-hydroxypropionic acid (3-HP) from lignocellulose. We rewired the central metabolism for the enhanced supply of acetyl-coenzyme A and nicotinamide adenine dinucleotide phosphate hydrogen, obtaining 30.0 g l-1 of FFAs from glucose, with productivity of up to 0.27 g l-1 h-1. Strengthening xylose uptake and catabolism promoted the synchronous utilization of glucose and xylose, which enabled the production of 38.2 g l-1 and 7.0 g l-1 FFAs from the glucose-xylose mixture and lignocellulosic hydrolysates, respectively. Finally, this efficient cell factory was metabolically transformed for 3-HP production with the highest titer of 79.6 g l-1 in fed-batch fermentation in mixed glucose and xylose.

Methanol biotransformation toward high-level production of fatty acid derivatives by engineering the industrial yeast Pichia pastoris.

Methanol-based biorefinery is a promising strategy to achieve carbon neutrality goals by linking CO2 capture and solar energy storage. As a typical methylotroph, Pichia pastoris shows great potential in methanol biotransformation. However, challenges still remain in engineering methanol metabolism for chemical overproduction. Here, we present the global rewiring of the central metabolism for efficient production of free fatty acids (FFAs; 23.4 g/L) from methanol, with an enhanced supply of precursors and cofactors, as well as decreased accumulation of formaldehyde. Finally, metabolic transforming of the fatty acid cell factory enabled overproduction of fatty alcohols (2.0 g/L) from methanol. This study demonstrated that global metabolic rewiring released the great potential of P. pastoris for methanol biotransformation toward chemical overproduction.

Identification of Embryonic Chicken Proteases Activating Newcastle Disease Virus and Their Roles in the Pathogenicity of Virus Used as In Ovo Vaccine.

In ovo vaccination is an attractive immunization approach for chickens. However, most live Newcastle disease virus (NDV) vaccine strains used safely after hatching are unsafe as in ovo vaccines due to their high pathogenicity for chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. Our previous studies reported that NDV strain TS09-C was a safe in ovo vaccine, and the F protein cleavage site (FCS) containing three basic amino acids (3B-FCS) was the crucial determinant of the attenuation of TS09-C in chicken embryos. Here, five trypsin-like proteases that activated NDV in chicken embryos were identified. The F protein with 3B-FCS was sensitive to the proteases Tmprss4, Tmprss9, and F7, was present in fewer tissue cells of chicken embryos, which limited the viral tropism, and was responsible for the attenuation of NDV with 3B-FCS, while the F protein with FCS containing two basic amino acids could be cleaved not only by Tmprss4, Tmprss9, and F7 but also by Prss23 and Cfd, was present in most tissue cells, and thereby was responsible for broad tissue tropism and high pathogenicity of virus in chicken embryos. Furthermore, when mixed with the protease inhibitors aprotinin and camostat, NDV with 2B-FCS exhibited greatly weakened pathogenicity in chicken embryos. Thus, our results extend the understanding of the molecular mechanism of NDV pathogenicity in chicken embryos and provide a novel molecular target for the rational design of in ovo vaccines, ensuring uniform and effective vaccine delivery and earlier induction of immune protection by the time of hatching. IMPORTANCE As an attractive immunization approach for chickens, in ovo vaccination can induce a considerable degree of protection by the time of hatching, provide support in closing the window in which birds are susceptible to infection, facilitate fast and uniform vaccine delivery, and reduce labor costs by the use of mechanized injectors. The commercial live Newcastle disease virus (NDV) vaccine strains are not safe for in ovo vaccination and cause the death of chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. In the present study, we identified five trypsin-like proteases that activate NDV in chicken embryos and elucidated their roles in the tissue tropism and pathogenicity of NDV used as in ovo vaccine. Finally, we revealed the molecular basis for the pathogenicity of NDV in chicken embryos and provided a novel strategy for the rational design of in ovo ND vaccines.

Spray vaccination with a Newcastle disease virus (NDV)-vectored infectious laryngotracheitis (ILT) vaccine protects commercial chickens from ILT in the presence of maternally-derived antibodies.

Infectious laryngotracheitis (ILT) poses a significant threat to the poultry industry, and vaccines play an important role in protection. However, due to the increasing scale of poultry production, there is an urgent need to develop vaccines that are suitable for convenient immunization methods such as spraying. Previous studies have shown that Newcastle disease virus (NDV)-ILT vaccines administered via intranasal and intraocular routes to commercial chickens carrying maternally-derived antibodies (MDAs) are still protective against ILT. In this study, a recombinant NDV (rNDV) was generated to express infectious laryngotracheitis virus (ILTV) glycoprotein B (gB), named rLS-gB, based on a full-length cDNA clone of the LaSota strain. The protective effect of different doses of rLS-gB administered by spray vaccination to commercial chickens at 1 d of age (doa) was evaluated. The chickens were exposed to 160-µm aerosol particles for 10 min for spray vaccination, and no adverse reactions were observed after vaccination. Despite the presence of anti-NDV MDAs and anti-ILTV MDAs in chickens, the ILTV- and NDV-specific antibody titres were significantly greater in the vaccinated groups than in the unvaccinated group. After challenge with a virulent ILTV strain, no clinical signs were observed in the 107 EID50/ml group compared to the other groups. Furthermore, vaccination with 107 EID50/ml rLS-gB significantly reduced the ILTV viral load and ameliorated gross and microscopic lesions in the trachea of chickens. Overall, these results suggested that rLS-gB is a safe and efficient candidate spray vaccine for ILT and is especially suitable for scaled chicken farms.

Manipulation of topoisomerase expression inhibits cell division but not growth and reveals a distinctive promoter structure in Synechocystis.

In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5'/3' gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.

C-terminal truncation of the hemagglutinin-neuraminidase (HN) protein enhances the virulence and immunogenicity of Newcastle disease virus (NDV) vaccine strain V4.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein with receptor recognition ability that plays an important role in the infection of cells by NDV. An alignment of NDV HN protein sequences of different genotypes showed that vaccine strains of NDV, such as the LaSota strain, generally have an HN protein of 577 amino acids. In comparison, the HN protein of the V4 strain has 616 amino acids, with 39 more amino acids at the C-terminus. In this study, we generated a recombinant NDV (rNDV) with a 39-amino-acid truncation at the HN C-terminus based on the full-length cDNA clone of the V4 strain. This rNDV, named rV4-HN-tr, displayed thermostability similar to that of the parental V4 strain. However, growth kinetics and pathogenicity analysis suggested that rV4-HN-tr is more virulent than the V4 strain. Notably, the C-terminus of HN affected the ability of the virus to adsorb onto cells. Structural predictions further suggested that the C-terminus of HN may obstruct the sialic acid binding site. Immunization of chickens with rV4-HN-tr induced a 3.5-fold higher level of NDV-specific antibodies than that obtained with the V4 strain and provided 100% immune protection against NDV challenge. Our study suggests that rV4-HN-tr is a thermostable, safe, and highly efficient vaccine candidate against Newcastle disease.

Engineering of formate dehydrogenase for improving conversion potential of carbon dioxide to formate.

Formate dehydrogenase (FDH) is a D-2-hydroxy acid dehydrogenase, which can reversibly reduce CO2 to formate and thus act as non-photosynthetic CO2 reductase. In order to increase catalytic efficiency of formate dehydrogenase for CO2 reduction, two mutants V328I/F285W and V354G/F285W were obtained of which reduction activity was about two times more than the parent CbFDHM2, and the formate production from CO2 catalyzed by mutants were 2.9 and 2.7-fold higher than that of the parent CbFDHM2. The mutants had greater potential in CO2 reduction. The optimal temperature for V328I/F285W and V354G/F285W was 55 °C, and they showed increasement of relative activity under 45 °C to 55 °C compared with parent. The optimal pH for the mutants was 9.0, and they showed excellent stability in pH 4.0-11.5. The kcat/Km values of mutants were 1.75 times higher than that of the parent. Then the molecular basis for its improvement of biochemical characteristics were preliminarily elucidated by computer-aided methods. All of these results further established a solid foundation for molecular modification of formate dehydrogenase and CO2 reduction.

Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris.

BACKGROUND: The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair over non-homologous end joining (NHEJ), we expressed five exonucleases that were fused with the Cas9 for enhancing end resection of double strand breaks (DSBs) of DNA cuts. RESULTS: The endogenous exonuclease Mre11 and Exo1 showed the highest positive rates in seamless deletion of FAA1, and fusing the MRE11 to the C-terminal of CAS9 had the highest positive rate and relatively high number of clones. We observed that expression of CAS9-MRE11 significantly improved positive rates when simultaneously seamless deletion of double genes (from 76.7 to 86.7%) and three genes (from 10.8 to 16.7%) when overexpressing RAD52. Furthermore, MRE11 overexpression significantly improved the genomic integration of multi-fragments with higher positive rate and clone number. CONCLUSIONS: Fusion expression of the endogenous exonuclease Mre11 with Cas9 enhances homologous recombination efficiency in P. pastoris. The strategy described here should facilitate the metabolic engineering of P. pastoris toward high-level production of value-added compounds.

Expanding the promoter toolbox for metabolic engineering of methylotrophic yeasts.

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. KEY POINTS: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha in metabolic engineeringare expounded. • Three promoter engineering strategies are introduced in order to expand the promoter toolbox.

Diagnosis of COVID-19 Pneumonia via a Novel Deep Learning Architecture.

COVID-19 is a contagious infection that has severe effects on the global economy and our daily life. Accurate diagnosis of COVID-19 is of importance for consultants, patients, and radiologists. In this study, we use the deep learning network AlexNet as the backbone, and enhance it with the following two aspects: 1) adding batch normalization to help accelerate the training, reducing the internal covariance shift; 2) replacing the fully connected layer in AlexNet with three classifiers: SNN, ELM, and RVFL. Therefore, we have three novel models from the deep COVID network (DC-Net) framework, which are named DC-Net-S, DC-Net-E, and DC-Net-R, respectively. After comparison, we find the proposed DC-Net-R achieves an average accuracy of 90.91% on a private dataset (available upon email request) comprising of 296 images while the specificity reaches 96.13%, and has the best performance among all three proposed classifiers. In addition, we show that our DC-Net-R also performs much better than other existing algorithms in the literature. Supplementary Information: The online version contains supplementary material available at 10.1007/s11390-020-0679-8.

Room-temperature two-dimensional plasmonic crystal semiconductor lasers.

Room-temperature plasmonic-crystal lasers have been demonstrated with a square-lattice gold nano-pillar arrays on top of InGaAs/GaAs quamtum wells on a GaAs substrate. The lasing wavelength is tunable in the range of 865-1001 nm by varying the lattice period. The lasers exhibit an extremely narrow linewidth and small divergence angle so could have great potential for various applications. An unexpected mirror cavity effect has been observed and investigated. The mirror-cavity lasers have a very low threshold and could be developed to realize electrically-driven plasmonic lasers.

Characterization of TCR+ and CD8+ head kidney leucocytes in Japanese flounder (Paralichthys olivaceus) with antisera against TCRα and CD8α.

T lymphocytes play an important role in cellular and adaptive immunity in vertebrates. The mechanisms of the fish immune system are little studied because of the lack of population-specific antibodies. This study examined the expression of two T lymphocyte markers, TCRα (PoTCRα) and CD8α (PoCD8α) in the Japanese flounder (Paralichthys olivaceus). The expression of PoTCRα and PoCD8α was mainly detected in immune/mucosal tissues. Recombinant PoTCRα and PoCD8α were expressed in pET32a and pET259, respectively. Then, rabbit anti-PoTCRα serum and rat anti-PoCD8α serum were prepared. Using serum, the characteristics of TCR+ and CD8+ head kidney leucocytes (HKLs) were investigated. The results of laser scanning confocal microscopy (LSCM) demonstrated that TCRα and CD8α were transmembrane proteins localized on the cell surface. The populations of CD8α- , CD8α+ , TCRα- , and TCRα+ were sorted by flow cytometry (FCM) and analysed using qRT-PCR. The results demonstrated that all TCRα+ /TCRα- or CD8α+ /CD8α- HKLs expressed IFN-γ. The CD4-1 and IgM transcripts were detected only in TCRα- and CD8α- cells. Furthermore, HKL mitogenesis was induced with concanavalin A (ConA) stimulation. Taken together, the results from LSCM and FCM analyses showed that mammalian and P. olivaceus TCR+ and CD8+ leucocytes share basic characteristics.

FTO promotes SREBP1c maturation and enhances CIDEC transcription during lipid accumulation in HepG2 cells.

The fat mass and obesity-associated (FTO) gene is tightly related to body weight and fat mass, and plays a pivotal role in regulating lipid accumulation in hepatocytes. However, the mechanisms underlying its function are poorly understood. Sterol regulatory element binding protein-1c (SREBP1c) is a transcription factor that regulates lipogenesis. Cell death-inducing DFFA (DNA fragmentation factor-α)-like effector c (CIDEC) plays a crucial role in lipid droplets (LDs) size controlling and lipid accumulation. In this report, we first observed that FTO overexpression in HepG2 cells resulted in an increase of lipogenesis and up-regulation of SREBP1c and CIDEC, two key regulatory factors in lipogenesis. In contrast, FTO knockdown in HepG2 cells resulted in a decrease of lipogenesis and down-regulation of SREBP1c and CIDEC expression. Moreover, SREBP1c knockdown resulted in a decrease of lipogenesis in HepG2 cells with FTO overexpression. In addition, FTO demethylation defect mutant presented less transcription of the key genes, and less nuclear translocation and maturation of SREBP1c. Further investigation demonstrated that overexpression of SREBP1c in HepG2 cells also promoted high CIDEC expression. Luciferase reporter assays showed that SREBP1c significantly stimulated CIDEC gene promoter activity. Finally, CIDEC knockdown reduced SREBP1c-induced lipogenesis. In conclusion, our studies suggest that FTO increased the lipid accumulation in hepatocytes by increasing nuclear translocation of SREBP1c and SREBP1c maturation, thus improving the transcriptional activity of LD-associated protein CIDEC. Our studies may provide new mechanistic insight into nonalcoholic fatty liver disease (NAFLD) mediated by FTO.

Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX.

Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways. We identified the essential phosphate acyltransferase enzyme PlsX (slr1510) as a key node in C18 fatty acyl-ACP consumption, repression of slr1510 increased octadecanol productivity threefold over the base strain and gave the highest specific titers reported for this host, 10.3mgg-1 DCW. PlsX catalyzes the first committed step of phosphatidic acid synthesis, and has not been characterized in Synechocystis previously. We found that accumulation of fatty alcohols impaired growth, altered the membrane composition, and caused a build-up of reactive oxygen species.

Pichia nanzhaoensis sp. nov. and Pichia paraexigua f.a., sp. nov., two yeast species isolated from rotting wood.

Four yeast strains were isolated from rotting wood samples collected in the Baotianman Nature Reserve in Henan Province, Central China. On the basis of sequence analysis of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer regions, they were suggested to be two novel species of the genus Pichia. Pichia nanzhaoensis sp. nov. produces one to four spherical ascospores per ascus, and is most closely related to Candida pseudolambica. Pichia paraexigua f.a., sp. nov. is a sister taxa to Pichia exigua, but the formation of ascospores was not observed on various sporulation media. P. nanzhaoensis sp. nov. can weakly assimilate inulin, whereas P. paraexigua sp. nov. can weakly assimilate d-glucosamine. The type strain of Pichia nanzhaoensis is NYNU 178136T (=CICC 33279T=CBS 15346T) and the type strain of Pichia paraexigua is NYNU 178135T (=CICC 33278T=CBS 15237T).

Reversible Cleavage/Formation of the Chromium-Chromium Quintuple Bond in the Highly Regioselective Alkyne Cyclotrimerization.

Herein we report the employment of the quintuply bonded dichromium amidinates [Cr{κ2 -HC(N-2,6-i Pr2 C6 H3 )(N-2,6-R2 C6 H3 )}]2 (R=iPr (1), Me (7)) as catalysts to mediate the [2+2+2] cyclotrimerization of terminal alkynes giving 1,3,5-trisubstituted benzenes. During the catalysis, the ultrashort Cr-Cr quintuple bond underwent reversible cleavage/formation, corroborated by the characterization of two inverted arene sandwich dichromium complexes (µ-η6 :η6 -1,3,5-(Me3 Si)3 C6 H3 )[Cr{κ2 -HC(N-2,6-i Pr2 C6 H3 )(N-2,6-R2 C6 H3 )}]2 (R=i Pr (5), Me (8)). In the presence of σ donors, such as THF and 2,4,6-Me3 C6 H2 CN, the bridging arene 1,3,5-(Me3 Si)3 C6 H3 in 5 and 8 was extruded and 1 and 7 were regenerated. Theoretical calculations were employed to disclose the reaction pathways of these highly regioselective [2+2+2] cylcotrimerization reactions of terminal alkynes.

[Effects of Combined Stress of High Density Polyethylene Microplastics and Chlorimuron-ethyl on Soybean Growth and Rhizosphere Bacterial Community].

With the vigorous development of agriculture in China， plastic mulch film and pesticides are widely used in agricultural production. However， the accumulation of microplastics （formed by the degradation of plastic mulch film） and pesticides in soil has also caused many environmental problems. At present， the environmental biological effects of microplastics or pesticides have been reported， but there are few studies on the combined effects on crop growth and the rhizosphere soil bacterial community. Therefore， in this study， the high density polyethylene microplastics （HDPE， 500 mesh） were designed to be co-treated with sulfonylurea herbicide chlorimuron-ethyl to study their effects on soybean growth. In addition， the effects of the combined stress of HDPE and chlorimuron-ethyl on soybean rhizosphere soil bacterial community diversity， structure composition， microbial community network， and soil function were investigated using high-throughput sequencing technology， interaction network， and PICRUSt2 function analysis to clarify the combined toxicity of HDPE and chlorimuron-ethyl to soybean. The results showed that the half-life of chlorimuron-ethyl in soil was prolonged by the 1% HDPE treatment （from 11.5 d to 14.3 d）， and the combined stress of HDPE and chlorimuron-ethyl had more obvious inhibition effects on soybean growth than that of the single pollutant or control. The HiSeq 2 500 sequencing showed that the rhizosphere bacterial community of soybean was composed of 20 phyla and 312 genera under combined stress， the number of phyla and genera was significantly less than that of the control and single pollutant treatment， and the relative abundances of bacteria with potential biological control and plant growth-promoting characteristics （such as Nocardioides and Sphingomonas） were reduced. Alpha diversity analysis showed that the combined stress significantly reduced the richness and diversity of the soybean rhizosphere bacterial community， and Beta diversity analysis showed that the combined stress significantly changed the structure of the bacterial community. The dominant flora of the rhizosphere bacterial community were regulated， and the abundances of secondary functional layers such as amino acid metabolism， energy metabolism， and lipid metabolism were reduced under combined stress by the analysis of LEfSe and PICRUSt2. It was inferred from the network analysis that the combined stress of HDPE and chlorimuron-ethyl reduced the total number of connections and network density of soil bacteria， simplified the network structure， and changed the important flora species to maintain the stability of the network. The results above indicated that the combined stress of HDPE and chlorimuron-ethyl significantly affected the growth of soybean and changed the rhizosphere bacterial community structure， soil function， and network structure. Compared with that of the single pollutant treatment， the potential risk of combined stress was greater. The results of this study can provide guidance for evaluating the ecological risks of polyethylene microplastics and chlorimuron-ethyl and for the remediation of contaminated soil.

Engineering Escherichia coli for high-yielding 2,5-Dimethylpyrazine synthesis from L-Threonine by reconstructing metabolic pathways and enhancing cofactors regeneration.

2,5-Dimethylpyrazine (2,5-DMP) is important pharmaceutical raw material and food flavoring agent. Recently, engineering microbes to produce 2,5-DMP has become an attractive alternative to chemical synthesis approach. In this study, metabolic engineering strategies were used to optimize the modified Escherichia coli BL21 (DE3) strain for efficient synthesis of 2,5-DMP using L-threonine dehydrogenase (EcTDH) from Escherichia coli BL21, NADH oxidase (EhNOX) from Enterococcus hirae, aminoacetone oxidase (ScAAO) from Streptococcus cristatus and L-threonine transporter protein (EcSstT) from Escherichia coli BL21, respectively. We further optimized the reaction conditions for synthesizing 2,5-DMP. In optimized conditions, the modified strain can convert L-threonine to obtain 2,5-DMP with a yield of 2897.30 mg/L. Therefore, the strategies used in this study contribute to the development of high-level cell factories for 2,5-DMP.

Cerebellar atrophy in genetic epileptic encephalopathies: A cohort study and a systematic review.

OBJECTIVE: To analyze cerebellar atrophy in genetic epileptic encephalopathies (EEs). METHODS: This research included a retrospective cohort study conducted from January 2016 to December 2023 and a systematic review on cerebellar atrophy in genetic EEs. Pediatric individuals who were diagnosed with EEs based on electroclinical features, carried causative gene variants, and exhibited cerebellar atrophy were recruited. Electroclinical features, neuroimaging findings, and causative variants of eligible individuals were analyzed. RESULTS: The cohort study showed 10 of 67 pediatric individuals (10/67; 15 %) who were diagnosed with genetic EEs had cerebellar atrophy; and 6 of the 10 individuals (6/10; 60 %) exhibited cerebellar signs. Diagnostic delay between the detection of cerebellar atrophy and the identification of genetic diagnosis existed in 6 individuals (6/10; 60 %) and the median duration was 4.4 years. A total of 32 genes, including 31 genes from the literature review and a newly identified SCN2A gene in this cohort, were reported associated with cerebellar atrophy in genetic EEs. Twenty-six genes (26/32; 81 %) accounted for cerebellar atrophy associated with other brain anomalies and 6 genes (6/32; 19 %) caused isolated cerebellar atrophy. Twenty-five genes (25/32; 78 %) showed late-onset cerebellar atrophy identified after the age of 1 year old. CONCLUSION: Cerebellar atrophy is not uncommon in genetic EEs and may serve as an indicator for molecular diagnosis in clinical practice. To shorten the diagnostic delay, follow-up neuroimaging study is crucial because of high rate of clinico-radiological dissociation and late-onset cerebellar atrophy in this patient group.

Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases.

Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12-C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.