Construction and Optimization of Nonclassical Isoprenoid Biosynthetic Pathways in Yeast Peroxisomes for (+)-Valencene Production.

Isoprenoids are a kind of natural product with various activities, but their plant extraction suffers low concentration. The rapid development of synthetic biology offers a sustainable route for supply of high-value-added natural products by engineering microorganisms. However, the complexity of cellular metabolism makes engineering endogenous isoprenoid biosynthetic pathways with metabolic interaction difficult. Here, for the first time, we constructed and optimized three types of isoprenoid pathways (the Haloarchaea-type, Thermoplasma-type, and isoprenoid alcohol pathway) in yeast peroxisomes for the synthesis of sesquiterpene (+)-valencene. In yeast, the Haloarchaea-type MVA pathway is more effective than the classical MVA pathway. MVK and IPK were determined to be the rate-limiting steps of the Haloarchaea-type MVA pathway, and the production of 869 mg/L (+)-valencene under fed-batch fermentation in shake flasks was realized. This work expands isoprenoid synthesis in eukaryotes and provides a more efficient pathway for isoprenoid synthesis.

Spatial-temporal regulation of fatty alcohol biosynthesis in yeast.

BACKGROUND: Construction of efficient microbial cell factories is one of the core steps for establishing green bio-manufacturing processes. However, the complex metabolic regulation makes it challenging in driving the metabolic flux toward the product biosynthesis. Dynamically coupling the biosynthetic pathways with the cellular metabolism at spatial-temporal manner should be helpful for improving the production with alleviating the cellular stresses. RESULTS: In this study, we observed the mismatch between fatty alcohol biosynthesis and cellular metabolism, which compromised the fatty alcohol production in Saccharomyces cerevisiae. To enhance the fatty alcohol production, we spatial-temporally regulated fatty alcohol biosynthetic pathway by peroxisomal compartmentalization (spatial) and dynamic regulation of gene expression (temporal). In particular, fatty acid/acyl-CoA responsive promoters were identified by comparative transcriptional analysis, which helped to dynamically regulate the expression of acyl-CoA reductase gene MaFAR1 and improved fatty alcohol biosynthesis by 1.62-fold. Furthermore, enhancing the peroxisomal supply of acyl-CoA and NADPH further improved fatty alcohol production to 282 mg/L, 2.52 times higher than the starting strain. CONCLUSIONS: This spatial-temporal regulation strategy partially coordinated fatty alcohol biosynthesis with cellular metabolism including peroxisome biogenesis and precursor supply, which should be applied for production of other products in microbes.

Expanding the neutral sites for integrated gene expression in Saccharomyces cerevisiae.

Construction of efficient microbial cell factories always requires assembling biosynthetic pathways and rewiring cellular metabolism with overexpression of multiple genes. Genomic integration is considered to be helpful for stable gene expression in compared with the episomal plasmids. However, the limited availability of suitable loci hinders the extensive metabolic engineering. We here characterized 30 neutral sites in Saccharomyces cerevisiae genome that did not affect cellular fitness by using expression cassettes of green fluorescent protein (eGFP) and fatty acyl-CoA reductase (MaFAR1) with the aid of efficient CRISPR-Cas9 technique. We found that integration of gene expression cassettes to different genome loci resulted a varied GFP signal and fatty alcohol production, which showed that genomic loci could be used for tuning gene expression. The characterized set of neutral sites should be helpful for extensively metabolic engineering of S. cerevisiae for chemical production and other purposes.

A comprehensive texture feature analysis framework of renal cell carcinoma: pathological, prognostic, and genomic evaluation based on CT images.

OBJECTIVES: We tried to realize accurate pathological classification, assessment of prognosis, and genomic molecular typing of renal cell carcinoma by CT texture feature analysis. To determine whether CT texture features can perform accurate pathological classification and evaluation of prognosis and genomic characteristics in renal cell carcinoma. METHODS: Patients with renal cell carcinoma from five open-source cohorts were analyzed retrospectively in this study. These data were randomly split to train and test machine learning algorithms to segment the lesion, predict the histological subtype, tumor stage, and pathological grade. Dice coefficient and performance metrics such as accuracy and AUC were calculated to evaluate the segmentation and classification model. Quantitative decomposition of the predictive model was conducted to explore the contribution of each feature. Besides, survival analysis and the statistical correlation between CT texture features, pathological, and genomic signatures were investigated. RESULTS: A total of 569 enhanced CT images of 443 patients (mean age 59.4, 278 males) were included in the analysis. In the segmentation task, the mean dice coefficient was 0.96 for the kidney and 0.88 for the cancer region. For classification of histologic subtype, tumor stage, and pathological grade, the model was on a par with radiologists and the AUC was 0.83 [Formula: see text] 0.1, 0.80 [Formula: see text] 0.1, and 0.77 [Formula: see text] 0.1 at 95% confidence intervals, respectively. Moreover, specific quantitative CT features related to clinical prognosis were identified. A strong statistical correlation (R2 = 0.83) between the feature crosses and genomic characteristics was shown. The structural equation modeling confirmed significant associations between CT features, pathological (ß = - 0.75), and molecular subtype (ß = - 0.30). CONCLUSIONS: The framework illustrates high performance in the pathological classification of renal cell carcinoma. Prognosis and genomic characteristics can be inferred by quantitative image analysis. KEY POINTS: • The analytical framework exhibits high-performance pathological classification of renal cell carcinoma and is on a par with human radiologists. • Quantitative decomposition of the predictive model shows that specific texture features contribute to histologic subtype and tumor stage classification. • Structural equation modeling shows the associations of genomic characteristics to CT texture features. Overall survival and molecular characteristics can be inferred by quantitative CT texture analysis in renal cell carcinoma.

Pan-cancer analysis of ASB3 and the potential clinical implications for immune microenvironment of glioblastoma multiforme.

Background: Ankyrin repeat and SOCS Box containing 3 (ASB3) is an E3 ubiquitin ligase. It has been reported to regulate the progression of some cancers, but no systematic pan-cancer analysis has been conducted to explore its function in prognosis and immune microenvironment. Method: In this study, mRNA expression data were downloaded from TCGA and GTEx database. Next generation sequencing data from 14 glioblastoma multiforme (GBM) samples by neurosurgical resection were used as validation dataset. Multiple bioinformatics methods (ssGSEA, Kaplan-Meier, Cox regression analysis, GSEA and online tools) were applied to explore ASB3 expression, gene activity, prognosis of patients in various cancers, and its correlation with clinical information, immune microenvironment and pertinent signal pathways in GBM. The biological function of ASB3 in tumor-infiltrating lymphocytes (TILs) was verified using an animal model. Results: We found that ASB3 was aberrant expressed in a variety of tumors, especially in GBM, and significantly correlated with the prognosis of cancer patients. The level of ASB3 was related to the TMB, MSI and immune cell infiltration in some cancer types. ASB3 had a negative association with immune infiltration and TME, including regulatory T cells (Tregs), cancer-associated fibroblasts, immunosuppressors and related signaling pathways in GBM. ASB3 overexpression reduced the proportion of Tregs in TILs. GSEA and PPI analysis also showed negative correlation between ASB3 expression and oncogenetic signaling pathways in GBM. Conclusion: A comprehensive pan-cancer analysis of ASB3 showed its potential function as a biomarker of cancer prognosis and effective prediction of immunotherapy response. This study not only enriches the understanding of the biological function of ASB3 in pan-cancer, especially in GBM immunity, but also provides a new reference for the personalized immunotherapy of GBM.

Biosynthesis of actarit using engineered Escherichia coli.

Actarit is widely regarded as a safe and effective drug for the treatment of rheumatoid arthritis. There is no report on the bioproductin of actarit so far. In this study, we demonstrated for the first time the development of an artificial actarit biosynthetic pathway in Escherichia coli. First, 4-aminophenylacetic acid is selected as precursor substrates for the production of actarit. Second, an N-acetyltransferase that can efficiently catalyse the esterification of acetyl-CoA and 4-aminophenylacetic acid to form actarit was discovered. Subsequently, an engineered E. coli that allows production of actarit from simple carbon sources was established. Finally, we further increased the production of actarit to 206 ± 16.9 mg/L by overexpression of shikimate dehydrogenase ydiB and shikimate kinase aroK.

Development of an artificial biosynthetic pathway for biosynthesis of (S)-reticuline based on HpaBC in engineered Escherichia coli.

Benzylisoquinoline alkaloids (BIAs) are an important class of plant secondary metabolites with a variety of pharmacological activities. Although they are widely used, traditionally these compounds are extracted from natural sources because their structure is too complicated to achieve economically feasible chemical synthesis. Thus, microbial biosynthesis of BIAs is expected to reduce dependence on natural extracts. (S)-Reticuline is an important precursor for BIAs biosynthesis. Therefore, it is an attractive engineering target. In this study, we reported the development of a novel (S)-reticuline biosynthetic pathway based on 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) in Escherichia coli. Then, we further improved the (S)-reticuline production to 307 ± 26.8 mg/L by increasing the availability of the precursor 3, 4-dihydroxyphenylacetaldehyde. The E. coli cell factory developed in this study can be used as a potential platform for further efficient biosynthesis of BIAs derivatives.

De novo biosynthesis of linalool from glucose in engineered Escherichia coli.

Linalool is an important terpenoids of floral scents and has wide applications. In the past, several groups reported on a strategy to establish biosynthesis of linalool in yeast based on co-expression of Saccharomyces cerevisiae farnesyl diphosphate synthase ERG20 and Actinidia arguta linalool synthase LIS. However, ERG20 has both geranyl diphosphate synthase and farnesyl diphosphate synthase activities, which can lead to metabolic flow to farnesyl diphosphate. In this study, a heterologous linalool biosynthesis pathway was constructed in Escherichia coli and showed that using Abies grandis geranyl diphosphate synthase GPPS2 instead of ERG20 can effectively improve linalool biosynthesis. Subsequently, we further improved the biosynthesis of linalool by overexpression of isopentenyl diphosphate isomerase Idi.

Transcriptomics of Planococcus kocurii O516 reveals the degrading metabolism of sulfamethoxazole in marine aquaculture wastewater.

Environmental threat induced by residual antibiotics in marine aquaculture wastewater is an urgent problem to be solved. In this study, one sulfamethoxazole (SMX)-degrading bacterium, Planococcus kocurii O516 was isolated from high SMX marine aquafarm. The isolate was able to consume more than 60% of SMX with the initial concentration of 10 mg L-1 within 72 h. Transcriptome analysis found great gene expression differences in the strains with or without SMX dosage. Three putatively differentially expressed proteins, namely AbrB/MazE/SpoVT family DNA-binding domain-containing protein, pantoate-beta-alanine ligase and MerR family transcriptional regulator, were annotated in detail. They were inferred to trigger the strain's response to SMX stress. Reverse transcription-quantitative PCR (RT-qPCR) analysis of four significantly different expressed genes accorded well with expression changes revealed by transcriptomics and confirmed the validity of transcriptome analysis. According to functional annotations of the proteins obtained by transcriptome sequencing and structural analysis of the intermediate metabolites by GC-MS, a possible SMX degradation pathway was reasonably proposed. SMX was first decomposed into sulfonamide and 5-methylisoxazole. The sulfonamide was then hydroxylated to form 4-(hydroxyamino) benzenesulfonamide. Subsequently, the sulfamic acid was detached, and 4-(hydroxyamino) phenol was formed. Finally, 4-aminophenol was generated from dehydroxylated of 4-(hydroxyamino) phenol. In sum, transcriptome analysis of the P. kocurii in response to SMX stress benefits to revealing the degradation pathway of SMX and will provide theoretical feasibility for the application of microbial method to treat the SMX-contaminated aquaculture wastewater.

De novo biosynthesis of 2-phenylethanol in engineered Pichia pastoris.

2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odor, which is widely used in cosmetics and food industries. Natural 2-PE is costly and cannot meet the market demand due to the relative low content of 2-PE in the plants. Thus, there is an increasing interest in the search for alternative routes for 2-PE production. Here we demonstrate the engineering of Pichia pastoris to produce 2-PE directly from simple sugars for the first time. We first demonstrated that improving downstream pathway from phenylpyruvate to 2-PE by overexpressing ARO10 and ADH6 could increase the biosynthesis of 2-PE. Then several genetic engineering strategies were developed to increase phenylpyruvate availability to improve 2-PE production. 1169 mg/L of 2-PE was accumulated in the final engineered strain. This study showed the potential of P. pastoris as a host strain to produce industrially interested 2-PE by metabolic engineering.

De Novo Biosynthesis of Indole-3-acetic Acid in Engineered Escherichia coli.

Indole-3-acetic acid (IAA) is considered the most common and important naturally occurring auxin in plants and a major regulator of plant growth and development. In this study, an aldehyde dehydrogenase AldH from Escherichia coli was found to convert indole-3-acetylaldehyde into IAA. Then we established an artificial pathway in engineered E. coli for microbial production of IAA from glucose. The overall pathway includes the upstream pathway from glucose to L-tryptophan and the downstream pathway from L-tryptophan to IAA. To our knowledge, this is the first report on the biosynthesis of IAA directly from a renewable carbon source. The study described here shows the way for the development of a beneficial microbe for biosynthesis of auxin and promoting plant growth in the future.

Biosynthesis of advanced biofuel farnesyl acetate using engineered Escherichia coli.

Diminishing petroleum reserves and the rapid accumulation of greenhouse gases lead to increasing interest in microbial biofuels. In this study, a heterologous farnesyl acetate biosynthesis pathway was constructed in Escherichia coli for the first time. Firstly, the AtoB, ERG13, tHMG1, ERG12, ERG8, MVD1, Idi, IspA and PgpB were expressed to accumulate farnesol in the E. coli cells. Then the alcohol acetyltransferase (ATF1) was heterologous overexpressed for the subsequent esterification farnesol to farnesyl acetate. The engineered strain DG 106 accumulated 128 ±â€¯10.5 mg/L of farnesyl acetate. Finally, the isopentenyl-diphosphate isomerase was further overexpressed, and the recombinant strain DG107 produced 201 ±â€¯11.7 mg/L of farnesyl acetate. This study shows the novel method for the biosynthesis of the advanced biofuel farnesyl acetate directly from glucose and highlight the enormous designing strategies for metabolic engineering of bioproducts.

Metabolic Engineering of Escherichia coli for Production of 2-Phenylethanol and 2-Phenylethyl Acetate from Glucose.

Rose-like odor 2-phenylethanol (2-PE) and its more fruit-like ester 2-phenylethyl acetate (2-PEAc) are two important aromatic compounds and have wide applications. In the past, 2-PE and 2-PEAc were mainly produced from l-phenylalanine. In this study, Escherichia coli was engineered to de novo biosynthesis of 2-PE and 2-PEAc from glucose: first, overexpression of deregulated 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase aroG fbr and chorismate mutase/prephenate dehydratase pheA fbr for increasing phenylpyruvate production in E. coli, subsequently, heterologous expression of decarboxylase kdc and overexpression of reductase yjgB for the conversion of phenylpyruvate to 2-PE, with the engineered strain DG01 producing 578 mg/L 2-PE, and, finally, heterologous expression of an aminotransferase aro8 to redirect the metabolic flux to phenylpyruvate. 2-PE (1016 mg/L) was accumulated in the engineered strain DG02. Alcohol acetyltransferase ATF1 from Saccharomyces cerevisiae can esterify a wide variety of alcohols, including 2-PE. We have further demonstrated the biosynthesis of 2-PEAc from glucose by overexpressing atf1 for the subsequent conversion of 2-PE to 2-PEAc. The engineered strain DG03 produced 687 mg/L 2-PEAc.