High-Temperature Tolerance Protein Engineering through Deep Evolution.

Protein engineering aimed at increasing temperature tolerance through iterative mutagenesis and high-throughput screening is often labor-intensive. Here, we developed a deep evolution (DeepEvo) strategy to engineer protein high-temperature tolerance by generating and selecting functional sequences using deep learning models. Drawing inspiration from the concept of evolution, we constructed a high-temperature tolerance selector based on a protein language model, acting as selective pressure in the high-dimensional latent spaces of protein sequences to enrich those with high-temperature tolerance. Simultaneously, we developed a variant generator using a generative adversarial network to produce protein sequence variants containing the desired function. Afterward, the iterative process involving the generator and selector was executed to accumulate high-temperature tolerance traits. We experimentally tested this approach on the model protein glyceraldehyde 3-phosphate dehydrogenase, obtaining 8 variants with high-temperature tolerance from just 30 generated sequences, achieving a success rate of over 26%, demonstrating the high efficiency of DeepEvo in engineering protein high-temperature tolerance.

Cyclization mechanism of monoterpenes catalyzed by monoterpene synthases in dipterocarpaceae.

Monoterpenoids are typically present in the secretory tissues of higher plants, and their biosynthesis is catalyzed by the action of monoterpene synthases (MTSs). However, the knowledge about these enzymes is restricted in a few plant species. MTSs are responsible for the complex cyclization of monoterpene precursors, resulting in the production of diverse monoterpene products. These enzymatic reactions are considered exceptionally complex in nature. Therefore, it is crucial to understand the catalytic mechanism of MTSs to elucidate their ability to produce diverse or specific monoterpenoid products. In our study, we analyzed thirteen genomes of Dipterocarpaceae and identified 38 MTSs that generate a variety of monoterpene products. By focusing on four MTSs with different product spectra and analyzing the formation mechanism of acyclic, monocyclic and bicyclic products in MTSs, we observed that even a single amino acid mutation can change the specificity and diversity of MTS products, which is due to the synergistic effect between the shape of the active cavity and the stabilization of carbon-positive intermediates that the mutation changing. Notably, residues N340, I448, and phosphoric acid groups were found to be significant contributors to the stabilization of intermediate terpinyl and pinene cations. Alterations in these residues, either directly or indirectly, can impact the synthesis of single monoterpenes or their mixtures. By revealing the role of key residues in the catalytic process and establishing the interaction model between specific residues and complex monoterpenes in MTSs, it will be possible to reasonably design and engineer different catalytic activities into existing MTSs, laying a foundation for the artificial design and industrial application of MTSs.

[Theoretical analysis and practical applications of the catalytic mechanism of flavonoid 6-hydroxylase].

Insufficient catalytic efficiency of flavonoid 6-hydroxylases in the fermentative production of scutellarin leads to the formation of at least about 18% of by-products. Here, the catalytic mechanisms of two flavonoid 6-hydroxylases, CYP82D4 and CYP706X, were investigated by molecular dynamics simulations and quantum chemical calculations. Our results show that CYP82D4 and CYP706X have almost identical energy barriers at the rate-determining step and thus similar reaction rates, while the relatively low substrate binding energy of CYP82D4 may facilitate product release, which is directly responsible for its higher catalytic efficiency. Based on the study of substrate entry and release processes, the catalytic efficiency of the L540A mutation of CYP82D4 increased by 1.37-fold, demonstrating the feasibility of theoretical calculations-guided engineering of flavonoid 6-hydroxylase. Overall, this study reveals the catalytic mechanism of flavonoid 6-hydroxylases, which may facilitate the modification and optimization of flavonoid 6-hydroxylases for efficient fermentative production of scutellarin.

Multilayered regulation of secondary metabolism in medicinal plants.

Medicinal plants represent a huge reservoir of secondary metabolites (SMs), substances with significant pharmaceutical and industrial potential. However, obtaining secondary metabolites remains a challenge due to their low-yield accumulation in medicinal plants; moreover, these secondary metabolites are produced through tightly coordinated pathways involving many spatiotemporally and environmentally regulated steps. The first regulatory layer involves a complex network of transcription factors; a second, more recently discovered layer of complexity in the regulation of SMs is epigenetic modification, such as DNA methylation, histone modification and small RNA-based mechanisms, which can jointly or separately influence secondary metabolites by regulating gene expression. Here, we summarize the findings in the fields of genetic and epigenetic regulation with a special emphasis on SMs in medicinal plants, providing a new perspective on the multiple layers of regulation of gene expression.

Construction of an artificial phosphoketolase pathway that efficiently catabolizes multiple carbon sources to acetyl-CoA.

The canonical glycolysis pathway is responsible for converting glucose into 2 molecules of acetyl-coenzyme A (acetyl-CoA) through a cascade of 11 biochemical reactions. Here, we have designed and constructed an artificial phosphoketolase (APK) pathway, which consists of only 3 types of biochemical reactions. The core enzyme in this pathway is phosphoketolase, while phosphatase and isomerase act as auxiliary enzymes. The APK pathway has the potential to achieve a 100% carbon yield to acetyl-CoA from any monosaccharide by integrating a one-carbon condensation reaction. We tested the APK pathway in vitro, demonstrating that it could efficiently catabolize typical C1-C6 carbohydrates to acetyl-CoA with yields ranging from 83% to 95%. Furthermore, we engineered Escherichia coli stain capable of growth utilizing APK pathway when glycerol act as a carbon source. This novel catabolic pathway holds promising route for future biomanufacturing and offering a stoichiometric production platform using multiple carbon sources.

Origin and evolution of the main starch biosynthetic enzymes.

Starch, a semi-crystalline energy storage form primarily found in plant plastids plays a crucial role in various food or no-food applications. Despite the starch biosynthetic pathway's main enzymes have been characterized, their origin and evolution remained a subject of debate. In this study, we conducted the comprehensive phylogenetic and structural analysis of three types of starch biosynthetic enzymes: starch synthase (SS), starch branching enzyme (SBE) and isoamylase-type debranching enzyme (ISA) from 51,151 annotated genomes. Our findings provide valuable insights into the possible scenario for the origin and evolution of the starch biosynthetic pathway. Initially, the ancestor of SBE can be traced back to an unidentified bacterium that existed before the formation of the last eukaryotic common ancestor (LECA) via horizontal gene transfer (HGT). This transfer event likely provided the eukaryote ancestor with the ability to synthesize glycogen. Furthermore, during the emergence of Archaeplastida, one clade of SS was transferred from Deltaproteobacteria by HGT, while ISA and the other clade of SS originated from Chlamydiae through endosymbiosis gene transfer (EGT). Both these transfer events collectively contributed to the establishment of the original starch biosynthetic pathway. Subsequently, after the divergence of Viridiplantae from Rhodophyta, all three enzymes underwent multiple duplications and N-terminus extension domain modifications, resulting in the formation of functionally specialized isoforms and ultimately leading to the complete starch biosynthetic pathway. By shedding light on the evolutionary origins of key enzymes involved in the starch biosynthetic pathway, this study provides important insights into the evolutionary events of plants.

Production of the antidepressant orcinol glucoside in Yarrowia lipolytica with yields over 6,400-fold higher than plant extraction.

Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.

Enzymatic Electrosynthesis of Glycine from CO2 and NH3.

Enzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2 . However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value-added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81 mM glycine was yielded with a highest reaction rate of 8.69 mg L-1 h-1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.

Transcriptome-referenced association study provides insights into the regulation of oil and fatty acid biosynthesis in Torreya grandis kernel.

INTRODUCTION: Torreya grandis is a gymnosperm belonging to Taxodiaceae. As an economically important tree, its kernels are edible and rich in oil with high unsaturated fatty acids, such as sciadonic acid. However, the kernels from different T. grandis landraces exhibit fatty acid and oil content variations. OBJECTIVES: As a gymnosperm, does T. grandis have special regulation mechanisms for oil biosynthesis? The aim of this study was to dissect the genetic architecture of fatty acid and oil content and the underlying mechanism in T. grandis. METHODS: We constructed a high integrity reference sequence of expressed regions of the genome in T. grandis and performed transcriptome-referenced association study (TRAS) for 10 fatty acid and oil traits of kernels in the 170 diverse T. grandis landraces. To confirm the TRAS result, we performed functional validation and molecular biology experiments for oil significantly associated genes. RESULTS: We identified 41 SNPs from 34 transcripts significantly associated with 7 traits by TRAS (-log10 (P) greater than 6.0). Results showed that LOB domain-containing protein 40 (LBD40) and surfeit locus protein 1 (SURF1) may be indirectly involved in the regulation of oil and sciadonic acid biosynthesis, respectively. Moreover, overexpression of TgLBD40 significantly increased seed oil content. The nonsynonymous variant in the TgLBD40 coding region discovered by TRAS could alter the oil content in plants. Pearson's correlation analysis and dual-luciferase assay indicated that TgLBD40 positively enhanced oil accumulation by affecting oil biosynthesis pathway genes, such as TgDGAT1. CONCLUSION: Our study provides new insights into the genetic basis of oil biosynthesis in T. grandis and demonstrates that integrating RNA sequencing and TRAS is a powerful strategy to perform association study independent of a reference genome for dissecting important traits in T. grandis.

Ethylene treatment promotes umami taste-active amino acids accumulation of Torreya grandis nuts post-harvest by comparative chemical and transcript analyses.

Amino acids play critical roles in physiological processes and also contribute significantly to fruit quality. In this study, the effect of exogenous ethylene on amino acids metabolism and related genes expression in Torreya grandis were investigated. The results revealed that ethylene treatment (3000 µL L-1 for 24 h) significantly increased amino acids level. Umami amino acids were distinctly upregulated in ethylene-treated versus control nuts, with glutamic and aspartic acids to demonstrate 1.9-fold and 2.1-fold increase. Transcriptome analysis revealed that deferentially expressed genes were mainly enriched in alanine aspartate and glutamate metabolism. RT-qPCR confirmed that ethylene treatment up-regulated expression of their biosynthesis genes (TgGOGAT1, TgAATC1, TgAATC4) concurrent with suppression of their degradation enzymes (TgGS2, TgGAD1, TgGAD3, TgASNS1). Ethylene treatment appears to promote umami taste-active amino acids and improve T. grandis nut quality post-harvest.

Thirteen Dipterocarpoideae genomes provide insights into their evolution and borneol biosynthesis.

Dipterocarpoideae, the largest subfamily of the Dipterocarpaceae, is a dominant component of Southeast Asian rainforests and is widely used as a source of wood, damar resin, medicine, and essential oil. However, many Dipterocarpoideae species are currently on the IUCN Red List owing to severe degradation of their habitats under global climate change and human disturbance. Genetic information regarding these taxa has only recently been reported with the sequencing of four Dipterocarp genomes, providing clues to the function and evolution of these species. Here, we report on 13 high-quality Dipterocarpoideae genome assemblies, ranging in size from 302.6 to 494.8 Mb and representing the five most species-rich genera in Dipterocarpoideae. Molecular dating analyses support the Western Gondwanaland origin of Dipterocarpaceae. Based on evolutionary analysis, we propose a three-step chromosome evolution scenario to describe the karyotypic evolution from an ancestor with six chromosomes to present-day species with 11 and 7 chromosomes. We discovered an expansion of genes encoding cellulose synthase (CesA), which is essential for cellulose biosynthesis and secondary cell-wall formation. We functionally identified five bornyl diphosphate synthase (BPPS) genes, which specifically catalyze the biosynthesis of borneol, a natural medicinal compound extracted from damar resin and oils, thus providing a basis for large-scale production of natural borneol in vitro.

Penalized logistic regressions with technical indicators predict up and down trends.

Correctly predicting up and down trends for stock prices is of immense important in the financial market. To further improve the prediction performance, in this paper we introduce five penalties: ridge, least absolute shrinkage and selection operator, elastic net, smoothly clipped absolute deviation and minimax concave penalty to logistic regressions with 19 technical indicators, and propose the five penalized logistic regressions to predict up and down trends for stock prices. Firstly, we translate the five penalized logistic log-likelihood functions into the five penalized weighted least squares functions and combine them with the tenfold cross-validation method to calculate the solution path to parameter estimators. Secondly, we combine the binomial deviation with cross-validation error as a risk measure to choose an appropriate tuning parameter for the penalty functions and apply the training set and the coordinate descent algorithm to obtain parameter estimators and probability estimators. Thirdly, we employ the testing set and the chosen optimal thresholds to construct two-class confusion matrices and receiver operating characteristic curves to assess the prediction performances to the five regressions. Finally, we compare the proposed five penalized logistic regressions with logistic regression, support vector machine and artificial neural network and found that the minimax concave penalty logistic regression performs the best in terms of the prediction performance to up and down trends for Google's stock prices. Therefore, in this paper we propose the five new prediction methods to improve the prediction accuracy of stock returns and bring economic benefits for investors.

Human epididymis protein 4 (HE4) is a novel immunohistochemical marker of neuroendocrine differentiation.

Human epididymis protein 4 (HE4) is originally described as an epididymis specific protein and now clinically used as a serum marker for ovarian carcinoma. However, the expression of HE4 in neuroendocrine neoplasms (NENs) has not been studied. By immunohistochemistry, the expressions of HE4 in 94 normal tissues and 484 NENs which included 242 well-differentiated NENs and 242 poorly differentiated NENs were studied. HE4 was positive in 90/94 (95.7%) of the neuroendocrine cells in normal tissues, 228/242 (94.2%) of well-differentiated NENs, and 206/242 (85.1%) of poorly differentiated NENs, and the expression of HE4 decreased progressively with loss of histological differentiation, with the positive rate of 96.2%, 92.7%, 92.3%, 85.4%, and 84.4% in NET-G1/carcinoid, NET-G2/atypical carcinoid, NET-G3, NEC-LC, and NEC-SC respectively. In NET-G1 and NET-G2, HE4 staining showed a peculiar polarized distribution, with an extraordinarily strong granular staining in subnuclear cytoplasm. A diffuse and uniform cytoplastic HE4 staining was observed in NET-G3 and poorly differentiated NENs. The positive rate of HE4 in primary tumors (91.1%, 387/425) was significantly higher than that of metastases (79.7%, 47/59) (p < 0.05). In a series of 70 pure non-NENs poorly differentiated carcinomas, the specificity rate of HE4 was 92.9% (65/70), which was in line with that of Syn. The negative rate of HE4 was 87.0% (40/46) in the non-neuroendocrine components of the MiNEN cases, which was lower than that of the pure non-neuroendocrine carcinomas (92.9%, 65/70) but without statistical significance (p > 0.05). HE4 may prove to be a useful immunohistochemical marker of neuroendocrine differentiation, although comparative studies and a more extensive analysis of other tissue types are necessary.

Metabolic engineering of Yarrowia lipolytica for scutellarin production.

Scutellarin related drugs have superior therapeutic effects on cerebrovascular and cardiovascular diseases. Here, an optimal biosynthetic pathway for scutellarin was constructed in Yarrowia lipolytica platform due to its excellent metabolic potential. By integrating multi-copies of core genes from different species, the production of scutellarin was increased from 15.11 mg/L to 94.79 mg/L and the ratio of scutellarin to the main by-product was improved about 110-fold in flask condition. Finally, the production of scutellarin was improved 23-fold and reached to 346 mg/L in fed-batch bioreactor, which was the highest reported titer for de novo production of scutellarin in microbes. Our results represent a solid basis for further production of natural products on unconventional yeasts and have a potential of industrial implementation.

The study of clinicopathologic features of cervical squamous carcinoma with invasive micropapillary like pattern and phenotype.

Invasive micropapillary carcinoma has been reported in the adenocarcinoma of many organs including cervix, and many studies have proved it has more invasive biological behavior. This study, for the first time, reports cervical squamous carcinoma with invasive micropapillary like pattern and phenotype (IMLPP) and further investigates its clinicopathologic features. Cervical squamous carcinoma with IMLPP was selected by histological characteristics and immunohistochemical staining. All patients' clinical information and pathological parameters were collected. Based on histological characteristics and immunohistochemical staining results, 24 cases, out of 104 cases of cervical squamous carcinoma, were identified as having invasive micropapillary like pattern. The staining of all 24 cases with EMA and MUC-1 showed the feature of "reverse polarity like". Meanwhile, patient age at diagnosis (P=0.011), maximum invasion depth (P=0.001), maximum diameter (P=0.015), lymphvascular space invasion (P<0.001), pelvic lymph node metastasis (P<0.001), metastasis (P=0.020), death (P=0.025) and FIGO stages (P=0.001) were related to the existence of IMLPP, independently of the proportion of IMLPP to the whole tumor in size. Univariate and multivariate disease-free survival analyses (follow-up time >12 months) showed significant statistical difference between cervical squamous carcinoma with or without IMLPP (P=0.016, P=0.043). Results from our study suggested that IMLPP may be associated with aggressive biological behavior in cervical squamous carcinoma. Therefore, pathologists should pay attention to the existence of it, no matter its proportion with relation to the whole tumor, and bring it to the attention of clinicians.

Five-year disease-free survival of Epstein-Barr virus-associated locoregionally advanced undifferentiated nasopharyngeal carcinoma patients treated with chemo-radiotherapy: a case report.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a tumor caused by epithelial cells covering the surface of the nasopharynx. NPC only accounted for less than 1% of all cancers diagnosed worldwide. However, the global incidence rates are highest in southern China. We report a case of local advanced undifferentiated NPC [specifically, vesicular nucleus cell carcinoma (VNCC) of NPC]. Long-term disease-free survival (DFS) of a patient with stage IVA NPC is reported. CASE DESCRIPTION: A 42-year-old male presented with a 4-month history of rhinorrhea and a lump in the left neck. The positron emission tomography (PET) showed local invasion to the surrounding tissues, specifically, the tumor invaded the brain. The pathological diagnosis was VNCC, the Epstein-Barr virus (EBV) was positive in tumor tissues by in situ hybridization. and the clinical diagnosis was stage IVA of NPC. The patient was treated with induction chemotherapy (IC) with gemcitabine and cisplatin (GP) followed by cisplatin/radiotherapy. The tumor lesions complete response (CR) after concurrent chemo-radiotherapy (CCRT). CONCLUSIONS: To date, the DFS time has been more than 5 years. IC with GP followed by CCRT should be the first choice of treatment for patients with locoregionally advanced NPC. In recent years, more and more studies have shown the efficacy of immunotherapy in treating recurrent or metastatic NPC patients, especially in patients or are programmed death-ligand 1 (PD-L1)-positive or have a high tumor mutation burden. In the future, immunotherapy may become a standard treatment in clinic and bring longer survival to patients.

[Artificial bioconversion of carbon dioxide].

Utilization of carbon dioxide (CO2) is a huge challenge for global sustainable development. Biological carbon fixation occurs in nature, but the low energy efficiency and slow speed hamper its commercialization. Physical-chemical carbon fixation is efficient, but relies on high energy consumption and often generates unwanted by-products. Combining the advantages of biological, physical and chemical technologies for efficient utilization of CO2 remains to be an urgent scientific and technological challenge to be addressed. Here, based on the development of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences in the past decade, we summarize the important progress in the design and construction of functional parts, pathways and systems for artificial bioconversion of carbon dioxide, including the breakthrough on the artificial synthesis of starch from CO2. Moreover, we prospect how to further develop the technologies for artificial bioconversion of carbon dioxide. These progress and perspectives provide new insight for achieving the goal of "carbon peaking and carbon neutrality".

[Advances in microbial synthesis of plant natural products].

Plant natural products are one of the main sources of small molecule drugs, nutraceuticals, cosmetics and fragrances, and play an important role in economy development. At present, the way of obtaining plant natural products mainly depends on direct extraction from plants, which is farm land occupying and time consuming. The contents of active ingredients in plants are usually low, and thus the production cost is high. By elucidating the biosynthetic pathways and reconstructing the pathways in microbial cells, plant natural products can be produced by fermentation using renewable raw materials. Microbial biosynthesis provides a new route for the supply of plant natural products. This review summarizes the research progress of microbial synthesis of terpenoids, flavonoids, phenylpropanoids and other important natural products of plants in Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Current research challenges and future prospects are also briefly discussed.