De novo biosynthesis of p-coumaric acid and caffeic acid from carboxymethyl-cellulose by microbial co-culture strategy.

BACKGROUND: Aromatic compounds, such as p-coumaric acid (p-CA) and caffeic acid, are secondary metabolites of various plants, and are widely used in diet and industry for their biological activities. In addition to expensive and unsustainable methods of plant extraction and chemical synthesis, the strategy for heterologous synthesis of aromatic compounds in microorganisms has received much attention. As the most abundant renewable resource in the world, lignocellulose is an economical and environmentally friendly alternative to edible, high-cost carbon sources such as glucose. RESULTS: In the present study, carboxymethyl-cellulose (CMC) was utilized as the sole carbon source, and a metabolically engineered Saccharomyces cerevisiae strain SK10-3 was co-cultured with other recombinant S. cerevisiae strains to achieve the bioconversion of value-added products from CMC. By optimizing the inoculation ratio, interval time, and carbon source content, the final titer of p-CA in 30 g/L CMC medium was increased to 71.71 mg/L, which was 155.9-fold higher than that achieved in mono-culture. The de novo biosynthesis of caffeic acid in the CMC medium was also achieved through a three-strain co-cultivation. Caffeic acid production was up to 16.91 mg/L after optimizing the inoculation ratio of these strains. CONCLUSION: De novo biosynthesis of p-CA and caffeic acid from lignocellulose through a co-cultivation strategy was achieved for the first time. This study provides favorable support for the biosynthesis of more high value-added products from economical substrates. In addition, the multi-strain co-culture strategy can effectively improve the final titer of the target products, which has high application potential in the field of industrial production.

High-copy genome integration and stable production of p-coumaric acid via a POT1-mediated strategy in Saccharomyces cerevisiae.

AIMS: To overcome the defective unstable production of p-coumaric acid (p-CA) using episomal plasmids and simultaneously achieve genetic stability and high-copy integration in Saccharomyces cerevisiae. METHODS AND RESULTS: Two-micron plasmids were used to obtain high titres of p-CA, but p-CA production was decreased significantly in a nonselective medium after 72 h. To overcome the defect of unstable p-CA production during fermentation, delta integration with the triosephosphate isomerase gene from Schizosaccharomyces pombe (POT1) was employed as a selection marker to integrate heterologous p-CA synthesis cassette, and the high-level p-CA-producing strain QT3-20 was identified. In shake flask fermentation, the final p-CA titre of QT3-20 reached 228.37 mg L-1 at 168 h, 11-fold higher than integrated strain QU3-20 using URA3 as the selective marker, and 9-fold higher than the best-performing episomal expression strain NKE1. Additionally, the p-CA titre and gene copy number remained stable after 100 generations of QT3-20 in a nonselective medium. CONCLUSION: We achieved high-copy genome integration and stable heterologous production of p-CA via a POT1-mediated strategy in S. cerevisiae. SIGNIFICANCE AND IMPACT OF STUDY: With superior genetic stability and production stability in a nonselective medium during fermentation, the high-level p-CA-producing strain constructed via POT1-mediated delta integration could serve as an efficient platform strain, to eliminate the threat of unstable and insufficient supply for future production of p-CA derivatives, make downstream processing and biosynthesis much simpler.

Comparative transcriptome analysis of genomic region deletion strain with enhanced L-tyrosine production in Saccharomyces cerevisiae.

OBJECTIVE: To determine the effect of large genomic region deletion in a Saccharomyces cerevisiae strain on tyrosine yield and to identify new genetic modification targets through transcriptome analysis. RESULTS: TAL was used to produce p-coumaric acid (p-CA) from tyrosine to quantity tyrosine yield. S. cerevisiae mutant strain NK14 with deletion of a 23.8 kb genomic region was identified to have p-CA production of 10.3 mg L- 1, while the wild-type strain BY4741 had p-CA production of 1.06 mg L- 1. Analysis of growth patterns and stress tolerance showed that the deletion did not affect the growth phenotype of NK14. Transcriptome analysis suggested that, compared to BY4741, genes related to glycolysis (ENO2, TKL1) and the tyrosine pathway (ARO1, ARO2, ARO4, ARO7, TYR1) were upregulated in NK14 at different levels. Besides genes related to the tyrosine biosynthetic pathway, amino acid transporters (AVT6, VBA5, THI72) and transcription factor (ARO80) also showed changes in transcription levels. CONCLUSIONS: We developed a strain with improved tyrosine yield and identified new genetic modification candidates for tyrosine production.

Metabolic engineering strategies for improvement of ethanol production in cellulolytic Saccharomyces cerevisiae.

As a traditional ethanol-producing microorganism, Saccharomyces cerevisiae is an ideal host for consolidated bioprocessing. However, expression of heterologous cellulase increases the metabolic burden in yeast, which results in low cellulase activity and poor cellulose degradation efficiency. In this study, cellulase-expressing yeast strains that could efficiently degrade different cellulosic substrates were created by optimizing cellulase ratios through a POT1-mediated δ-integration strategy. Metabolic engineering strategies, including optimization of codon usage, promoter and signal peptide, were also included in this system. We also confirmed that heterologous cellulase expression in cellulosic yeast induced the unfolded protein response. To enhance protein folding capacity, the endoplasmic reticulum chaperone protein BiP and the disulfide isomerase Pdi1p were overexpressed, and the Golgi membrane protein Ca2+/Mn2+ ATPase Pmr1p was disrupted to decrease the glycosylation of cellulase. The resultant strain, SK18-3, could produce 5.4 g L-1 ethanol with carboxymethyl-cellulose. Strain SK12-50 achieved 4.7 g L-1 ethanol production with phosphoric acid swollen cellulose hydrolysis. When Avicel was used as the substrate, 3.8 g L-1 ethanol (75% of the theoretical maximum yield) was produced in SK13-34. This work will significantly increase our knowledge of how to engineer optimal yeast strains for biofuel production from cellulosic biomass.

POT1-mediated δ-integration strategy for high-copy, stable expression of heterologous proteins in Saccharomyces cerevisiae.

In biotechnological industry, increased expression cassette stability and copy number serve as important means of maintaining consistently high production levels of heterologous proteins in Saccharomyces cerevisiae. In this study, we combined δ sequences for site-specific integration with TPI1 gene from Schizosaccharomyces pombe (POT1) as a selection marker to realize high-copy integration and stable expression of heterologous proteins in S. cerevisiae. With the newly developed POT1 platform, a 32-copy integration of enhanced green fluorescent protein (EGFP) expression cassette was obtained in a single round and was stably maintained after 100 generations of growth in a rich complex medium. Talaromyces emersonii cellobiohydrolase I gene was synthesized with S. cerevisiae codon bias and expressed under the control of TPI1 promoter and terminator via POT1-mediated δ-integration; the highest specific activity yielded 238 mU g-1 of dry cell weight when p-nitrophenyl-ß-D-cellobioside was used as substrate, whereas the highest activity in cellulose hydrolysis reached 67% Avicel conversion. POT1-mediated δ-integration produces high protein levels over a wide dynamic range and enables broad applications in metabolic engineering and synthetic biology.

Reduced production of ethyl carbamate for wine fermentation by deleting CAR1 in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.

[Advances in synthesis of 2-phenylethanol].

2-phenylethanol (2-PE), an aromatic alcohol with a rose fragrance, is the second most widely used flavoring substance in the world. It is widely used in the cosmetic, food, and pharmaceutical industries. This paper introduces the chemical synthesis methods of 2-PE and the synthetic pathways in plants and microorganisms, summarizes the strategies to improve the microbial synthesis of 2-PE, reviews the research progress in de novo synthesis of 2-PE in microorganisms, and makes an outlook on the research prospects, aiming to provide a theoretical basis for the industrial production of 2-PE.

Developing Multi-Copy Chromosomal Integration Strategies for Heterologous Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae.

Caffeic acid, a plant-sourced phenolic compound, has a variety of biological activities, such as antioxidant and antimicrobial properties. The caffeic acid biosynthetic pathway was initially constructed in S. cerevisiae, using codon-optimized TAL (coTAL, encoding tyrosine ammonia lyase) from Rhodobacter capsulatus, coC3H (encoding p-coumaric acid 3-hydroxylase) and coCPR1 (encoding cytochrome P450 reductase 1) from Arabidopsis thaliana in 2 µ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of coTAL via delta integration with the POT1 gene (encoding triose phosphate isomerase) as selection marker and episomal expression of coC3H, coCPR1 using the episomal plasmid pLC-c3 were combined, and caffeic acid production was proved to be improved. Next, the delta and rDNA multi-copy integration methods were applied to integrate the genes coC3H and coCPR1 into the chromosome of high p-coumaric acid yielding strain QT3-20. The strain D9 constructed via delta integration outperformed the other strains, leading to 50-fold increased caffeic acid production in optimized rich media compared with the initial construct. The intercomparison between three alternative multi-copy strategies for de novo synthesis of caffeic acid in S. cerevisiae suggested that delta-integration was effective in improving caffeic acid productivity, providing a promising strategy for the production of valuable bio-based chemicals in recombinant S. cerevisiae.

Quantitative Evaluation of Gastrocnemius Medialis Stiffness During Passive Stretching Using Shear Wave Elastography in Patients with Parkinson's Disease: A Prospective Preliminary Study.

OBJECTIVE: To prospectively investigate the feasibility of shear wave elastography (SWE) as a new quantitative and objective method for evaluating the stiffness of the gastrocnemius medialis (GM) muscle during passive stretching in patients with Parkinson's disease (PD). MATERIALS AND METHODS: SWE of the GM muscle was performed in 28 patients with PD [13 female and 15 male; mean age ± standard deviation (SD): 63.0 ± 8.5 years] and 12 healthy controls (5 female and 7 male; mean age ± SD: 59.3 ± 6.4 years) during passive ankle rotation. A Young's modulus-ankle angle curve was constructed. The GM slack angle and baseline Young's modulus (E0) were compared between the markedly symptomatic and mildly symptomatic sides of patients with PD, and healthy controls. Additionally, the correlation between the GM slack angle and the severity of rigidity, and the observer reproducibility of SWE in determining the GM slack angle were evaluated. RESULTS: The GM slack angle was smaller on both the markedly and mildly symptomatic sides in patients with PD than in healthy controls (mean ± SD of -29.13° ± 3.79° and -25.65° ± 3.39°, respectively, vs. -21.22° ± 3.52°; p < 0.001 and p = 0.006, respectively). Additionally, in patients with PD, the GM slack angle on the markedly symptomatic side was smaller than that on the mildly symptomatic side (p = 0.003). The E0 value was lower on both the markedly and mildly symptomatic sides in patients with PD than in healthy controls (mean ± SD of 10.11 ± 2.85 kPa and 10.08 ± 1.88 kPa, respectively, vs. 12.23 ± 1.02 kPa; p = 0.012 and p < 0.001, respectively). However, no significant difference was found between the markedly and mildly symptomatic sides in patients with PD (p = 0.634). A negative linear relationship was observed between the GM slack angle and lower limb rigidity score on the markedly symptomatic side in patients with PD (r = -0.719; p < 0.001). The intraclass correlation coefficients for observer reproducibility of SWE ranged from 0.880 to 0.951. CONCLUSION: The slack angle determined by SWE may be a useful quantitative and reproducible method for evaluating muscle stiffness in patients with PD.

De Novo Biosynthesis of Caffeic Acid from Glucose by Engineered Saccharomyces cerevisiae.

Caffeic acid is a plant phenolic compound possessing extensive pharmacological activities. Here, we identified that p-coumaric acid 3-hydroxylase from Arabidopsis thaliana was capable of hydroxylating p-coumaric acid to form caffeic acid in Saccharomyces cerevisiae. Then, we introduced a combined caffeic acid biosynthetic pathway into S. cerevisiae and obtained 0.183 mg L-1 caffeic acid from glucose. Next we improved the tyrosine biosynthesis in S. cerevisiae by blocking the pathway flux to aromatic alcohols and eliminating the tyrosine-induced feedback inhibition resulting in caffeic acid production of 2.780 mg L-1. Finally, the medium was optimized, and the highest caffeic acid production obtained was 11.432 mg L-1 in YPD medium containing 4% glucose. This study opens a route to produce caffeic acid from glucose in S. cerevisiae and establishes a platform for the biosynthesis of caffeic acid derived metabolites.

Optimization of the l-tyrosine metabolic pathway in Saccharomyces cerevisiae by analyzing p-coumaric acid production.

In this study, we applied a series of genetic modifications to wild-type S. cerevisiae strain BY4741 to address the bottlenecks in the l-tyrosine pathway. A tyrosine ammonia-lyase (TAL) gene from Rhodobacter capsulatus, which can catalyze conversion of l-tyrosine into p-coumaric acid, was overexpressed to facilitate the analysis of l-tyrosine and test the strain's capability to synthesize heterologous derivatives. First, we enhanced the supply of precursors by overexpressing transaldolase gene TAL1, enolase II gene ENO2, and pentafunctional enzyme gene ARO1 resulting in a 1.55-fold increase in p-coumaric acid production. Second, feedback inhibition of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase was relieved by overexpressing the mutated feedback-resistant ARO4 K229L and ARO7 G141S , and a 3.61-fold improvement of p-coumaric acid production was obtained. Finally, formation of byproducts was decreased by deleting pyruvate decarboxylase gene PDC5 and phenylpyruvate decarboxylase gene ARO10, and p-coumaric acid production was increased 2.52-fold. The best producer-when TAL1, ENO2, ARO1, ARO4 K229L , ARO7 G141S , and TAL were overexpressed, and PDC5 and ARO10 were deleted-increased p-coumaric acid production by 14.08-fold (from 1.4 to 19.71 mg L-1). Our study provided a valuable insight into the optimization of l-tyrosine metabolic pathway.

The Efficacy of Contrast Transthoracic Echocardiography and Contrast Transcranial Doppler for the Detection of Patent Foramen Ovale Related to Cryptogenic Stroke.

BACKGROUND: Patent foramen ovale (PFO) has been linked to the pathophysiology of cryptogenic stroke. Contrast transesophageal echocardiography (cTEE) is the current gold standard for PFO diagnosis, but it has the disadvantage of being semi-invasive and does not exempt from risks. As a diagnostic test, the efficacy of contrast transthoracic echocardiography (cTTE) and contrast transcranial Doppler (cTCD) is controversial. This study is aimed at investigating the efficacy of cTTE and cTCD versus cTEE in PFO detection, exploring a more cost-effective and reliable method for the diagnosis of PFO related to cryptogenic stroke. METHODS: From August 2019 to January 2020, a total of 213 patients with suspected PFO were included in our study. All patients underwent cTEE, cTCD, and cTTE examinations. cTTE3 was named for using a cutoff of 3 beats to detect PFO during cTTE, and cTTE5 represented a cutoff of 5 beats. A cutoff of cTCD grade III was named cTCD III. A cutoff of grade IV was named cTCD IV. cTTE3+cTCD IV was used for the combination of a cutoff of 3 beats during cTTE with grade IV of cTCD. cTTE5+cTCD III combined a cutoff of 5 beats during cTTE with cTCD grade III. Taking cTEE as the gold standard, we compared the sensitivity, specificity, negative likelihood ratio (-LR), and misdiagnosis rate for PFO detection among the above methods. RESULTS: A total of 161 of 213 (76%) patients had PFO confirmed by cTEE. With the spontaneous Valsalva maneuver, the sensitivity, specificity, negative likelihood ratio (-LR), and misdiagnosis rate of cTTE3 in PFO diagnosis were 60%, 90%, 44%, and 10%, respectively, and those for cTTE5 were 76%, 78%, 31% and 22%, respectively. The sensitivity, specificity, negative likelihood ratio (-LR), and misdiagnosis rate of cTCD III were 80%, 71%, 29%, and 29%, respectively, while those for cTCD IV were 55%, 90%, 49%, and 10%, respectively. When cTTE and cTCD were combined to diagnose PFO, the specificity and misdiagnosis rate were significantly improved, especially cTTE3+cTCD IV, with 100% specificity and a misdiagnosis rate of 0. CONCLUSION: cTTE or cTCD can be used for preliminary PFO related to cryptogenic stroke findings. The combination of the two methods can improve the specificity of PFO diagnosis, especially using the cutoff of cTTE3+cTCD IV.

A high-throughput method for screening of L-tyrosine high-yield strains by Saccharomyces cerevisiae.

A biosensor screening assay based on the synthesis of betaxanthin was applied to relatively high throughput screening of the L-tyrosine mutant library. In the assays, fluorescence output showed a linear relationship between extracellular L-tyrosine content and yellow pigment formation. In addition, the yellow pigment accumulation of the L-tyrosine high-yield strain can be easily distinguished with the naked eye compared with the wild-type strain. As a result, numerous mutants that exhibited significantly increased coloration, were screened out after random mutagenesis, and p-coumaric acid production in mutants NK-A3 and NK-B4, were remarkably improved by 4-fold more than that of the wild-type strain. In general, this study provides a novel strategy for screening mutant libraries in the search for highly L-tyrosine-producing strains.