Reprogramming Yeast Metabolism from Alcoholic Fermentation to Lipogenesis.

Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism's natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine-tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.

Peroxisomal metabolic coupling improves fatty alcohol production from sole methanol in yeast.

Methanol is an ideal feedstock for chemical and biological manufacturing. Constructing an efficient cell factory is essential for producing complex compounds through methanol biotransformation, in which coordinating methanol use and product synthesis is often necessary. In methylotrophic yeast, methanol utilization mainly occurs in peroxisomes, which creates challenges in driving the metabolic flux toward product biosynthesis. Here, we observed that constructing the cytosolic biosynthesis pathway resulted in compromised fatty alcohol production in the methylotrophic yeast Ogataea polymorpha. Alternatively, peroxisomal coupling of fatty alcohol biosynthesis and methanol utilization significantly improved fatty alcohol production by 3.9-fold. Enhancing the supply of precursor fatty acyl-CoA and cofactor NADPH in the peroxisomes by global metabolic rewiring further improved fatty alcohol production by 2.5-fold and produced 3.6 g/L fatty alcohols from methanol under fed-batch fermentation. We demonstrated that peroxisome compartmentalization is helpful for coupling methanol utilization and product synthesis, and with this approach, constructing efficient microbial cell factories for methanol biotransformation is feasible.

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.

Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast.

Advances in synthetic biology enable microbial hosts to synthesize valuable natural products in an efficient, cost-competitive and safe manner. However, current engineering endeavors focus mainly on enzyme engineering and pathway optimization, leaving the role of cofactors in microbial production of natural products and cofactor engineering largely ignored. Here we systematically engineered the supply and recycling of three cofactors (FADH2, S-adenosyl-L-methion and NADPH) in the yeast Saccharomyces cerevisiae, for high-level production of the phenolic acids caffeic acid and ferulic acid, the precursors of many pharmaceutical molecules. Tailored engineering strategies were developed for rewiring biosynthesis, compartmentalization and recycling of the cofactors, which enabled the highest production of caffeic acid (5.5 ± 0.2 g l-1) and ferulic acid (3.8 ± 0.3 g l-1) in microbial cell factories. These results demonstrate that cofactors play an essential role in driving natural product biosynthesis and the engineering strategies described here can be easily adopted for regulating the metabolism of other cofactors.

Abnormalities in spontaneous brain activity and functional connectivity are associated with cognitive impairments in children with type 1 diabetes mellitus.

BACKGROUND: It remains unclear whether alterations in brain function occur in the early stage of pediatric type 1 diabetes mellitus(T1DM). We aimed to examine changes in spontaneous brain activity and functional connectivity (FC) in children with T1DM using resting-state functional magnetic resonance imaging (rs-fMRI), and to pinpoint potential links between neural changes and cognitive performance. METHODS: In this study, 22 T1DM children and 21 age-, sex-matched healthy controls underwent rs-fMRI. The amplitude of low frequency fluctuations (ALFF) and seed-based FC analysis were performed to examine changes in intrinsic brain activity and functional networks in T1DM children. Partial correlation analyses were utilized to explore the correlations between ALFF values and clinical parameters. RESULTS: The ALFF values were significantly lower in the lingual gyrus (LG) and higher in the left medial superior frontal gyrus (MSFG) in T1DM children compared to controls. Subsequent FC analysis indicated that the LG had decreased FC with bilateral inferior occipital gyrus, and the left MSFG had decreased FC with right precentral gyrus, right inferior parietal gyrus and right postcentral gyrus in children with T1DM. The ALFF values of LG were positively correlated with full-scale intelligence quotient and age at disease onset in T1DM children, while the ALFF values of left MSFG were positively correlated with working memory scores. CONCLUSION: Our findings revealed abnormal spontaneous activity and FC in brain regions related to visual, memory, default mode network, and sensorimotor network in the early stage of T1DM children, which may aid in further understanding the mechanisms underlying T1DM-associated cognitive dysfunction.

Global metabolic rewiring of the nonconventional yeast Ogataea polymorpha for biosynthesis of the sesquiterpenoid ß-elemene.

Bioproduction of natural products via microbial cell factories is a promising alternative to traditional plant extraction. Recently, nonconventional microorganisms have emerged as attractive chassis hosts for biomanufacturing. One such microorganism, Ogataea polymorpha is an industrial yeast used for protein expression with numerous advantages, such as thermal-tolerance, a wide substrate spectrum and high-density fermentation. Here, we systematically rewired the cellular metabolism of O. polymorpha to achieve high-level production of the sesquiterpenoid ß-elemene by optimizing the mevalonate pathway, enhancing the supply of NADPH and acetyl-CoA, and downregulating competitive pathways. The engineered strain produced 509 mg/L and 4.7 g/L of ß-elemene under batch and fed-batch fermentation, respectively. Therefore, this study identified the potential industrial application of O. polymorpha as a good microbial platform for producing sesquiterpenoids.

Engineering yeast for high-level production of diterpenoid sclareol.

The diterpenoid sclareol is an industrially important precursor for alternative sustainable supply of ambergris. However, its current production from plant extraction is neither economical nor environmental-friendly, since it requires laborious and cost-intensive purification procedures and plants cultivation is susceptible to environmental factors. Engineering cell factories for bio-manufacturing can enable sustainable production of natural products. However, stringent metabolic regulation poses challenges to rewire cellular metabolism for overproduction of compounds of interest. Here we used a modular approach to globally rewire the cellular metabolism for improving sclareol production to 11.4 g/L in budding yeast Saccharomyces cerevisiae, the highest reported diterpenoid titer in microbes. Metabolic flux analysis showed that modular balanced metabolism drove the metabolic flux toward the biosynthesis of targeted molecules, and transcriptomic analysis revealed that the expression of central metabolism genes was shaped for a new balanced metabolism, which laid a foundation in extensive metabolic engineering of other microbial species for sustainable bio-production.

Altered gray matter volume in children with newly diagnosed type 1 diabetes mellitus.

BACKGROUND: Type 1 diabetes mellitus (T1DM) affects the development of cognitive function in children, which may be due to deficits in brain structures or functions. It is unclear whether children with T1DM experience alterations in the gray matter (GM) structure at the initial stages of the disease. This study investigated GM structure alterations in children with newly diagnosed T1DM. METHODS: Based on 3D T1-weighted MR images, we investigated the gray matter volume (GMV) of 35 newly diagnosed T1DM children and 35 age- and sex-matched healthy controls using voxel-based morphometry. The brain regions with significant differences in GMV between the newly diagnosed T1DM children and the controls were extracted and the correlation with clinical data was assessed. RESULTS: Compared with the control group, children with newly diagnosed T1DM had a lower GMV in the right inferior and middle temporal gyri, right lingual gyrus, and left superior frontal gyrus. In T1DM subjects, the GMV of the right middle temporal gyrus was positively correlated with IQ but was negatively correlated with HbA1c. CONCLUSIONS: Our findings provide compelling evidence that GM abnormalities occur during early disease stages in T1DM children, which may be a potential neurobiological mechanism underlying cognitive deficits. IMPACT: Using an efficient method to analyze gray matter changes in T1DM is very important. The anterior, posterior, and temporal brain regions are susceptible to T1DM in children. Recent glucose variability may affect regional gray matter volume in children with newly diagnosed T1DM. Structural changes were documented in the gray matter of the brain even at the early stages of the disease in children with T1DM.

Disruption of brain regional homogeneity and functional connectivity in male NAFLD: evidence from a pilot resting-state fMRI study.

BACKGROUND: The neurophysiological mechanisms underlying cognitive deficits in non-alcoholic fatty liver disease (NAFLD) remain unknown. Cognitive changes may be caused by brain alterations in neural activity and functional connectivity (FC). AIM: This study aims to investigate the alterations between spontaneous brain neural activity and FC in male NAFLD patients and the relationship of neural activity with cognitive performance. METHODS: In this prospective study, 33 male pre-cirrhosis NAFLD subjects and 20 male controls matched for age, education level, and body mass index. All participants underwent resting-state functional magnetic resonance imaging scans and neuropsychological examinations. Regional homogeneity (ReHo) analysis was used to investigate the brain function in NAFLD, and regions with significantly altered ReHo were selected as seeds for subsequent FC analysis. Partial correlation analysis was used to assess the relationships between altered ReHo measures and cognitive performance indicators. RESULTS: Compared with the controls, the NAFLD patients showed increased ReHo in the opercular part of the right inferior frontal gyrus (IFGoperc) and decreased ReHo in the right middle frontal gyrus (MFG) and left superior parietal gyrus (SPG). The subsequent FC analysis showed increased FC between these regions (right IFGoperc, right MFG, and left SPG) and nodes of the default mode network (DMN) (such as left supraMarginal, left median cingulate and paracingulate gyri, left precuneus, orbital part of left medial frontal gyrus, and bilateral posterior cingulate gyrus). In addition, significant positive correlations were observed between NAFLD patients' clock drawing test scores and altered ReHo in prefrontal cortices (right IFGoperc and right MFG). CONCLUSION: Before developing cirrhosis, NAFLD patients showed altered neural activity in several brain regions and altered FC between the salience network and DMN. These alterations could potentially be a compensatory mechanism to maintain cognitive function in pre-cirrhosis NAFLD patients.

Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris.

The industrial yeast Pichia pastoris has been harnessed extensively for production of proteins, and it is attracting attention as a chassis cell factory for production of chemicals. However, the lack of synthetic biology tools makes it challenging in rewiring P. pastoris metabolism. We here extensively engineered the recombination machinery by establishing a CRISPR-Cas9 based genome editing platform, which improved the homologous recombination (HR) efficiency by more than 54 times, in particular, enhanced the simultaneously assembly of multiple fragments by 13.5 times. We also found that the key HR-relating gene RAD52 of P. pastoris was largely repressed in compared to that of Saccharomyces cerevisiae. This gene editing system enabled efficient seamless gene disruption, genome integration and multiple gene assembly with positive rates of 68-90%. With this efficient genome editing platform, we characterized 46 potential genome integration sites and 18 promoters at different growth conditions. This library of neutral sites and promoters enabled two-factorial regulation of gene expression and metabolic pathways and resulted in a 30-fold range of fatty alcohol production (12.6-380 mg/l). The expanding genetic toolbox will facilitate extensive rewiring of P. pastoris for chemical production, and also shed light on engineering of other non-conventional yeasts.

Dual-Polarized Multi-Beam Fixed-Frequency Beam Scanning Leaky-Wave Antenna.

A fixed-frequency beam-scanning leaky-wave antenna (LWA) array with three switchable dual-polarized beams is proposed and experimentally demonstrated. The proposed LWA array consists of three groups of spoof surface plasmon polaritons (SPPs) LWAs with different modulation period lengths and a control circuit. Each group of SPPs LWAs can independently control the beam steering at a fixed frequency by loading varactor diodes. The proposed antenna can be configured in both multi-beam mode and single-beam mode, where the multi-beam mode with optional two or three dual-polarized beams. The beam width can be flexibly adjusted from narrow to wide by switching between multi-beam and single-beam states. The prototype of the proposed LWA array is fabricated and measured, and both simulation and experimental results show that the antenna can accomplish a fixed frequency beam scanning at an operating frequency of 3.3 to 3.8 GHz with a maximum scanning range of about 35° in multi-beam mode and about 55° in single-beam mode. It could be a promising candidate for application in the space-air-ground integrated network scenario in satellite communication and future 6G communication systems.

Skeleton-Reorganizing Coupling Reactions of Cycloheptatriene and Cycloalkenones with Amines.

Skeletal reorganization reactions have emerged as an intriguing tool for converting readily available compounds into complicated molecules inaccessible by traditional methods. Herein, we report a unique skeleton-reorganizing coupling reaction of cycloheptatriene and cycloalkenones with amines. In the presence of Rh/acid catalysis, cycloheptatriene can selectively couple with anilines to deliver fused 1,2-dihydroquinoline products. Mechanistic studies indicate that the retro-Mannich type ring-opening and subsequent intramolecular Povarov reaction account for the ring reorganization. Our mechanistic studies also revealed that skeleton-reorganizing amination between anilines and cycloalkenones can be achieved with acid. The synthetic utilization of this skeleton-reorganizing coupling reaction was showcased with a gram-scale reaction, synthetic derivatizations, and the late-stage modification of commercial drugs.

CT-based radiomics for differentiating intracranial contrast extravasation from intraparenchymal haemorrhage after mechanical thrombectomy.

OBJECTIVE: To develop a nonenhanced CT-based radiomic signature for the differentiation of iodinated contrast extravasation from intraparenchymal haemorrhage (IPH) following mechanical thrombectomy. METHODS: Patients diagnosed with acute ischaemic stroke who underwent mechanical thrombectomy in 4 institutions from December 2017 to June 2020 were included in this retrospective study. The study population was divided into a training cohort and a validation cohort. The nonenhanced CT images taken after mechanical thrombectomy were used to extract radiomic features. The maximum relevance minimum redundancy (mRMR) algorithm was used to eliminate confounding variables. Afterwards, least absolute shrinkage and selection operator (LASSO) logistic regression was used to generate the radiomic signature. The diagnostic performance of the radiomic signature was evaluated by the area under the curve (AUC), accuracy, specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV). RESULTS: A total of 166 intraparenchymal areas of hyperattenuation from 101 patients were used. The areas of hyperattenuation were randomly allocated to the training and validation cohorts at a ratio of 7:3. The AUC of the radiomic signature was 0.848 (95% confidence interval (CI) 0.780-0.917) in the training cohort and 0.826 (95% CI 0.705-0.948) in the validation cohort. The accuracy of the radiomic signature was 77.6%, with a sensitivity of 76.7%, a specificity of 78.9%, a PPV of 85.2%, and a NPV of 68.2% in the validation cohort. CONCLUSIONS: The radiomic signature constructed based on initial post-operative nonenhanced CT after mechanical thrombectomy can effectively differentiate IPH from iodinated contrast extravasation. KEY POINTS: • Radiomic features were extracted from intraparenchymal areas of hyperattenuation on initial post-operative CT scans after mechanical thrombectomy. • The nonenhanced CT-based radiomic signature can differentiate IPH from iodinated contrast extravasation early. • The radiomic signature may help prevent unnecessary rescanning after mechanical thrombectomy, especially in cases where contrast extravasation is highly suggestive.

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.

Characterizing methanol metabolism-related promoters for metabolic engineering of Ogataea polymorpha.

Promoters play an important role in regulating gene expression, and construction of microbial cell factories requires multiple promoters for balancing the metabolic pathways. However, there are only a limited number of characterized promoters for gene expression in the methylotrophic yeast Ogataea polymorpha, which hampers the extensive harnessing of this important yeast toward a cell factory. Here we characterized the promoters of methanol utilization pathway, precursor supply pathway, and reactive oxygen species (ROS) defense system, by using a green fluorescence protein variant (GFPUV) as a quantification signal. Finally, the characterized promoters were used for tuning a fatty alcohol biosynthetic pathway in O. polymorpha and realized fatty alcohol production from methanol. This promoter box should be helpful for gene expression and pathway optimization in the methylotrophic yeast O. polymorpha. KEY POINTS : • 22 promoters related to methanol metabolism were characterized in O. polymorpha. • Promoter truncation resulted shorter and compact promoters. • Promoters with various strengths were used for regulating a fatty alcohol biosynthesis from methanol.

Grain chalkiness traits is affected by the synthesis and dynamic accumulation of the storage protein in rice.

BACKGROUOND: Grain chalkiness lowers the market value of rice. Alleviating grain chalkiness is the most challenging issue in many rice-producing areas of the world. Nitrogen (N) metabolism has received increasing attention as a result of its relationship with grain chalkiness, although little information is available on the mechanism of N-induced grain chalk. RESULTS: A highly chalky rice variety OM052 was used to explore the protein synthesis and its accumulation in the grain exposed to N topdressing (N+) at the panicle initiation stage and a control (N-). The results showed that chalky kernels were stimulated by the N+ treatment and more prone to occur on the top and primary rachis. The grain protein content was increased because of the increased average and maximum rates of protein accumulation during grain filling, which was related to the enhanced activities of glutamine synthetase, glutamate synthase, glutamic oxalo-acetic transaminase and glutamate pyruvate transaminase under the N+ treatment. The activities of these enzymes at 15 days after flowering (DAF) were notably positively correlated with grain chalky traits and protein content. CONCLUSION: N topdressing regulates the synthesis and accumulation of the protein by affecting the key enzymes, especially at 15 DAF, which is attributed to grain chalkiness in rice. © 2021 Society of Chemical Industry.

Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways enables high-level production of miltiradiene in yeast.

Miltiradiene is a key intermediate in the biosynthesis of many important natural diterpene compounds with significant pharmacological activity, including triptolide, tanshinones, carnosic acid and carnosol. Sufficient accumulation of miltiradiene is vital for the production of these medicinal compounds. In this study, comprehensive engineering strategies were applied to construct a high-yielding miltiradiene producing yeast strain. First, a chassis strain that can accumulate 2.1 g L-1 geranylgeraniol was constructed. Then, diterpene synthases from various species were evaluated for their ability to produce miltiradiene, and a chimeric miltiradiene synthase, consisting of class II diterpene synthase (di-TPS) CfTPS1 from Coleus forskohlii (Plectranthus barbatus) and class I di-TPS SmKSL1 from Salvia miltiorrhiza showed the highest efficiency in the conversion of GGPP to miltiradiene in yeast. Moreover, the miltiradiene yield was further improved by protein modification, which resulted in a final yield of 550.7 mg L-1 in shake flasks and 3.5 g L-1 in a 5-L bioreactor. This work offers an efficient and green process for the production of the important intermediate miltiradiene, and lays a foundation for further pathway reconstruction and the biotechnological production of valuable natural diterpenes.

Efficient targeted mutation of genomic essential genes in yeast Saccharomyces cerevisiae.

Targeted gene mutation by allelic replacement is important for functional genomic analysis and metabolic engineering. However, it is challenging in mutating the essential genes with the traditional method by using a selection marker, since the first step of essential gene knockout will result in a lethal phenotype. Here, we developed a two-end selection marker (Two-ESM) method for site-directed mutation of essential genes in Saccharomyces cerevisiae with the aid of the CRISPR/Cas9 system. With this method, single and double mutations of the essential gene ERG20 (encoding farnesyl diphosphate synthase) in S. cerevisiae were successfully constructed with high efficiencies of 100%. In addition, the Two-ESM method significantly improved the mutation efficiency and simplified the genetic manipulation procedure compared with traditional methods. The genome integration and mutation efficiencies were further improved by dynamic regulation of mutant gene expression and optimization of the integration modules. This Two-ESM method will facilitate the construction of genomic mutations of essential genes for functional genomic analysis and metabolic flux regulation in yeasts. KEY POINTS: • A Two-ESM strategy achieves mutations of essential genes with high efficiency of 100%. • The optimized three-module method improves the integration efficiency by more than three times. • This method will facilitate the functional genomic analysis and metabolic flux regulation.