Production of Camellia oleifera Abel Seed Oil for Injection: Extraction, Analysis, Deacidification, Decolorization, and Deodorization.

Camellia seed oil (CSO), as a nutrient-rich edible oil, is widely used in foods, cosmetics, and other fields. In this work, the extraction, deacidification, decolorization, and deodorization processes of CSO were respectively optimized for meeting injectable oil standards. The results showed that the CSO extraction rate reached the highest level of 94% at optimized conditions (ultrasonic time, 31.2 min; reaction pH, 9.2; and reaction time, 3.5 h). The physicochemical indexes of CSO and 10 other vegetable oils were evaluated by the principal component analysis method, and the overall scores of vegetable oils were ranked as camellia seed oil > olive oil > rice oil > peanut oil > sesame oil > corn oil > soybean oil > sunflower oil > rapeseed oil > walnut oil > flaxseed oil. The physicochemical indicators of CSO were the most ideal among the 11 vegetable oils, which means that CSO is suitable as an injectable oil. Through the optimized processes of the deacidification, decolorization, and deodorization, the CSO acid value was reduced to 0.0515 mg KOH/g, the decolorization rate reached a maximum of 93.86%, and the OD430 was 0.015, meeting the requirement (≤0.045 of OD430) of injectable oil. After the deodorization process, these parameters of the refractive index, acid value, saponification value, iodine value, absorbance, unsaponifiable, moisture and volatiles, fatty acid composition, and heavy metal limits all met the pharmacopoeia standards of injectable oil in many countries and regions. The possibility of CSO as an injectable oil was first verified through refining-process optimization and nutritional index analysis, providing an important technical reference for the high-value utilization of vegetable oil.

Effects of Extraction Strategies on Yield, Physicochemical and Antioxidant Properties of Pumpkin Seed Oil.

This study investigated the effects of three extraction methods, including cold pressing (CP), microwave pretreatment pressing (MP), and supercritical fluid extraction (SFE), on the yield, physicochemical properties, bioactive compounds content, and antioxidant properties of pumpkin seed oil (PSO). Furthermore, the correlation between bioactive compounds and the antioxidant properties of PSO was determined. The results revealed that the yield of PSO extracted using the three methods was in the order of SFE > MP > CP. Additionally, the PSO generated by SFE showed the highest unsaturated fatty acid content, followed by MP and CP. Additionally, MP-PSO exhibited the highest acid value and saponification value, while SFE-PSO displayed the highest moisture content, peroxide value, and iodine value. Moreover, the PSO generated by MP demonstrated superior antioxidant properties compared to that of PSOs from CP and SFE in the oxidation induction, DPPH, FRAP, and ABTS tests. Finally, the correlation analysis revealed that specific types of bioactive compounds, such as ß-sitosterol and γ-tocopherol, were highly correlated with the antioxidant properties of PSOs. Consequently, this study provides comprehensive knowledge regarding PSO extraction, physicochemical properties, bioactive compound extraction, and the correlated antioxidant properties.

Selection of Salt-Tolerance and Ester-Producing Mutant Saccharomyces cerevisiae to Improve Flavour Formation of Soy Sauce during Co-Fermentation with Torulopsis globosa.

Soy sauce, as a traditional seasoning, is widely favoured by Chinese and other Asian people for its unique colour, smell, and taste. In this study, a salt-tolerance Saccharomyces cerevisiae strain HF-130 was obtained via three rounds of ARTP (Atmospheric and Room Temperature Plasma) mutagenesis and high-salt based screening. The ethanol production of mutant HF-130 was increased by 98.8% in very high gravity fermentation. Furthermore, ATF1 gene was overexpressed in strain HF-130, generating ester-producing strain HF-130-ATF1. The ethyl acetate concentration of strain HF-130-ATF1 was increased by 130% compared to the strain HF-130. Finally, the soy sauce fermentation performance of Torulopsis globosa and HF-130-ATF1 was compared with T. globosa, HF-130, HF-130-ATF1, and Torulopsis and HF-130. Results showed ethyl acetate and isoamyl acetate concentrations in co-fermentation of T. globosa and HF-130-ATF1 were increased by 2.8-fold and 3.3-fold, respectively. In addition, the concentrations of ethyl propionate, ethyl caprylate, phenylethyl acetate, ethyl caprate, isobutyl acetate, isoamyl alcohol, phenylethyl alcohol, and phenylacetaldehyde were also improved. Notably, other three important flavour components, trimethylsilyl decyl ester, 2-methylbutanol, and octanoic acid were also detected in the co-fermentation of T. globosa and HF-130-ATF1, but not detected in the control strain T. globosa. This work is of great significance for improving the traditional soy sauce fermentation mode, and thus improving the flavour formation of soy sauce.

Screening and Remodeling of Enone Oxidoreductase for High Production of 2(or 5)-Ethyl-5(or 2)-methyl-4-hydroxy-3(2H)-Furanone in Saccharomyces Cerevisiae.

Owing to its unique fragrance, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) is widely used as a food flavoring agent and has high demand. Enone oxidoreductase is a vital enzyme involved in HEMF production. In this study, an enone oxidoreductase from Naumovozyma dairenensis CBS 421 (NDEO) was used for HEMF production for the first time. The mutant NDEOT183W,K290W was obtained through semirational protein engineering, which increased the HEMF yield by 75.2%. Finally, the engineered strain BM4 produced the highest HEMF yield, 194.42 mg L-1 in 132 h. Our study revealed that HEMF production can be improved in Saccharomyces cerevisiae and that this is an efficient method to improve the activity of enone oxidoreductase, which is important for the industrial synthesis of furanone.

Development and characterization of a glycine biosensor system for fine-tuned metabolic regulation in Escherichia coli.

BACKGROUND: In vivo biosensors have a wide range of applications, ranging from the detection of metabolites to the regulation of metabolic networks, providing versatile tools for synthetic biology and metabolic engineering. However, in view of the vast array of metabolite molecules, the existing number and performance of biosensors is far from sufficient, limiting their potential applications in metabolic engineering. Therefore, we developed the synthetic glycine-ON and -OFF riboswitches for metabolic regulation and directed evolution of enzyme in Escherichia coli. RESULTS: The results showed that a synthetic glycine-OFF riboswitch (glyOFF6) and an increased-detection-range synthetic glycine-ON riboswitch (glyON14) were successfully screened from a library based on the Bacillus subtilis glycine riboswitch using fluorescence-activated cell sorting (FACS) and tetA-based dual genetic selection. The two synthetic glycine riboswitches were successfully used in tunable regulation of lactate synthesis, dynamic regulation of serine synthesis and directed evolution of alanine-glyoxylate aminotransferase in Escherichia coli, respectively. Mutants AGXT22 and AGXT26 of alanine-glyoxylate aminotransferase with an increase of 58% and 73% enzyme activity were obtained by using a high-throughput screening platform based on the synthetic glycine-OFF riboswitch, and successfully used to increase the 5-aminolevulinic acid yield of engineered Escherichia coli. CONCLUSIONS: A synthetic glycine-OFF riboswitch and an increased-detection-range synthetic glycine-ON riboswitch were successfully designed and screened. The developed riboswitches showed broad application in tunable regulation, dynamic regulation and directed evolution of enzyme in E. coli.

Natural 5-Aminolevulinic Acid: Sources, Biosynthesis, Detection and Applications.

5-Aminolevulinic acid (5-ALA) is the key precursor for the biosynthesis of tetrapyrrole compounds, with wide applications in medicine, agriculture and other burgeoning fields. Because of its potential applications and disadvantages of chemical synthesis, alternative biotechnological methods have drawn increasing attention. In this review, the recent progress in biosynthetic pathways and regulatory mechanisms of 5-ALA synthesis in biological hosts are summarized. The research progress on 5-ALA biosynthesis via the C4/C5 pathway in microbial cells is emphasized, and the corresponding biotechnological design strategies are highlighted and discussed in detail. In addition, the detection methods and applications of 5-ALA are also reviewed. Finally, perspectives on potential strategies for improving the biosynthesis of 5-ALA and understanding the related mechanisms to further promote its industrial application are conceived and proposed.

Cell Catalysis of Citrate to Itaconate by Engineered Halomonas bluephagenesis.

Itaconic acid (IA), an important five-carbon unsaturated dicarboxylic acid, is one of the top 12 renewable chemicals with an urgent need to reduce industrial production costs. Halomonas bluephagenesis, which possesses the potential for cost-effective bioproduction of chemicals and organic acids due to its ability to grow under open nonsterile conditions and high tolerance to organic acid salts, was genetically engineered and used to produce IA from citrate by a cell catalytic strategy. Here, two essential genes (cis-aconitate decarboxylase encoding gene cadA and aconitase (ACN) encoding gene acn) were introduced into H. bluephagenesis to construct an IA biosynthesis pathway. Further engineering modifications including coexpression of molecular chaperones GroESL, increasing the copy number of the gene encoding rate-limiting enzyme ACN, and weakening the competing pathway were implemented. Under the optimized condition for the cell catalytic system, the engineered strain TAZI-08 produced 451.45 mM (58.73 g/L) IA from 500 mM citrate, with 93.24% conversion in 36 h and a productivity of 1.63 g/(L h). An intermittent feeding strategy further increased the IA titer to 488.86 mM (63.60 g/L). The IA titer and citrate conversion in H. bluephagenesis are the highest among heterologous hosts reported so far, demonstrating that this strain is a suitable chassis for hyperproduction of IA.

GAT1 Gene, the GATA Transcription Activator, Regulates the Production of Higher Alcohol during Wheat Beer Fermentation by Saccharomyces cerevisiae.

Uncoordinated carbon-nitrogen ratio in raw materials will lead to excessive contents of higher alcohols in alcoholic beverages. The effect of GAT1 gene, the GATA transcription activator, on higher alcohol biosynthesis was investigated to clarify the mechanism of Saccharomyces cerevisiae regulating higher alcohol metabolism under high concentrations of free amino nitrogen (FAN). The availability of FAN by strain SDT1K with a GAT1 double-copy deletion was 28.31% lower than that of parent strain S17, and the yield of higher alcohols was 33.91% lower. The transcript levels of the downstream target genes of GAT1 and higher alcohol production in the double-copy deletion mutant suggested that a part of the effect of GAT1 deletion on higher alcohol production was the downregulation of GAP1, ARO9, and ARO10. This study shows that GATA factors can effectively regulate the metabolism of higher alcohols in S. cerevisiae and provides valuable insights into higher alcohol biosynthesis, showing great significance for the wheat beer industry.

Genetic Diversity for Accelerating Microbial Adaptive Laboratory Evolution.

Adaptive laboratory evolution (ALE) is a widely used and highly effective tool for improving microbial phenotypes and investigating the evolutionary roots of biological phenomena. Serving as the raw materials of evolution, mutations have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions. The generation of genetic diversity is therefore a core technology for accelerating ALE, and a high-quality mutant library is crucial to its success. Because of its importance, technologies for generating genetic diversity have undergone rapid development in recent years. Here, we review the existing techniques for the construction of mutant libraries, briefly introduce their mechanisms and applications, discuss ongoing and emerging efforts to apply engineering technologies in the construction of mutant libraries, and suggest future perspectives for library construction.

Advances in the Extraction, Purification and Detection of the Natural Product 1-Deoxynojirimycin.

1-Deoxynojirimycin (1-DNJ), a polyhydroxylated alkaloid, is a highly selective and potent glycosidase inhibitor that has garnered great interest as a tool to study cellular recognition and as a potential therapeutic agent. The development of analytical methods for the quantification polyhydroxylated alkaloids in natural products requires a multifaceted approach. Many publications over the past five decades have described analytical methods for this compound. However, recently more advanced techniques have come to prominence for sample extraction, purification, detection, and identification. This review provides an updated, extensive overview of the available methods for the extraction, purification, identification or detection of 1-DNJ. The review highlights different strategies for the design of 1-DNJ detection methods, which we analyzed in light of recent detection data. Finally, we conclude with perspectives on possible strategies for increasing the efficiency of identification and quantification of 1-DNJ in the future.

Increased Acetate Ester Production of Polyploid Industrial Brewer's Yeast Strains via Precise and Seamless "Self-cloning" Integration Strategy.

BACKGROUND: Enhancing the industrial yeast strains ethyl acetate yield through a precise and seamless genetic manipulation strategy without any extraneous DNA sequences is an essential requisite and significant demand. OBJECTIVES: For increasing the ethyl acetate yield of industrial brewer's yeast strain, all the ATF1 alleles were overexpressed through "self-cloning" integration strategy. MATERIAL AND METHODS: Escherichia coli strain DH5α was utilized for plasmid construction. ATF1 alleles were overexpressed through a precise and seamless insertion of the PGK1 promoter in industrial brewer's yeast strain S6. In addition, growth rates, ATF1 mRNA levels, AATase activity, the fermentation performance of the engineered strains, and gas chromatography (GC) analysis was conducted. RESULTS: The two engineered strains (S6-P-12 and S6-P-30) overexpressed all ATF1 alleles but unaffected normal growth. The ATF1 mRNA levels of the S6-P-12 and S6-P-30 were all 4-fold higher than that of S6. The AATase (Alcohol acetyl transferases, encoded by ATF1 gene) activity of the two engineered strains was all 3-fold higher than that of the parent strain. In the beer fermentation at 10 ℃, the concentrations of ethyl acetate produced by the engineered strains S6-P-12 and S6-P-30 was increased to 23.98 and 24.00 mg L-1, respectively, about 20.44% and 20.54% higher than that of S6. CONCLUSIONS: These results verify that the ethyl acetate yield could be enhanced by the overexpressed of ATF1 in the polyploid industrial brewer's yeast strains via "self-cloning" integration strategy. The present study provides a reference for target gene modification in the diploid or polyploid industrial yeast strains.

Identification by comparative transcriptomics of core regulatory genes for higher alcohol production in a top-fermenting yeast at different temperatures in beer fermentation.

Undesirable flavor caused by excessive higher alcohols restrains the development of the wheat beer industry. To clarify the regulation mechanism of the metabolism of higher alcohols in wheat beer brewing by the top-fermenting yeast Saccharomyces cerevisiae S17, the effect of temperature on the fermentation performance and transcriptional levels of relevant genes was investigated. The strain S17 produced 297.85 mg/L of higher alcohols at 20 °C, and the production did not increase at 25 °C, reaching about 297.43 mg/L. Metabolite analysis and transcriptome sequencing showed that the metabolic pathways of branched-chain amino acids, pyruvate, phenylalanine, and proline were the decisive factors that affected the formation of higher alcohols. Fourteen most promising genes were selected to evaluate the effects of single-gene deletions on the synthesis of higher alcohols. The total production of higher alcohols by the mutants Δtir1 and Δgap1 was reduced by 23.5 and 19.66% compared with the parent strain S17, respectively. The results confirmed that TIR1 and GAP1 are crucial regulatory genes in the metabolism of higher alcohols in the top-fermenting yeast. This study provides valuable knowledge on the metabolic pathways of higher alcohols and new strategies for reducing the amounts of higher alcohols in wheat beer.

Modulating acetate ester and higher alcohol production in Saccharomyces cerevisiae through the cofactor engineering.

Flavor production by esters or by higher alcohols play a key role in the sensorial quality of fermented alcoholic beverages. In Saccharomyces cerevisiae cells, the syntheses of esters and higher alcohols are considerably influenced by intracellular CoA levels catalyzed by pantothenate kinase. In this work, we examined the effects of cofactor CoA and acetyl-CoA synthesis on the metabolism of esters and higher alcohols. Strains 12α-BAP2 and 12α+ATF1 where generated by deleting and overexpressing BAP2 (encoded branched-chain amino acid permease) and ATF1 (encoded alcohol acetyl transferases), respectively, in the parent 12α strains. Then, 12α-BAP2+CAB1 and 12α-BAP2+CAB3 strains were obtained by overexpressing CAB1 (encoded pantothenate kinase Cab1) and CAB3 (encoded pantothenate kinase Cab3) in the 12α-BAP2 strain, and 12α-BAP2+CAB1+ATF1 and 12α-BAP2+CAB3+ATF1 were generated by overexpressing ATF1 in the pantothenate kinase overexpression strains. The acetate ester level in 12α-BAP2 was slightly changed relative to that in the control strain 12α, whereas the acetate ester levels in 12α-BAP2+CAB1, 12α-BAP2+CAB3, 12α-BAP2+CAB1+ATF1, and 12α-BAP2+CAB3+ATF1 were distinctly increased (44-118% for ethyl acetate and 18-57% for isoamyl acetate). The levels of n-propanol, methyl-1-butanol, isopentanol, isobutanol, and phenethylol levels were changed and varied among the six engineered strains. The levels of acetate esters and higher alcohols can be modulated by changing the CoA and acetyl-CoA levels. The method proposed in this work supplies a practical means of breeding yeast strains by modulating acetate ester and higher alcohol production.

Recent advances in CRISPR/Cas9 mediated genome editing in Bacillus subtilis.

Genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used in various host cells. Its widespread adoption has been largely developed by the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR) system, which uses an easily customizable specificity RNA-guided DNA endonuclease, such as Cas9. Recently, CRISPR/Cas9 mediated genome engineering has been widely applied to model organisms, including Bacillus subtilis, enabling facile, rapid high-fidelity modification of endogenous native genes. Here, we reviewed the recent progress in B. subtilis gene editing using CRISPR/Cas9 based tools, and highlighted state-of-the-art strategies for design of CRISPR/Cas9 system. Finally, future perspectives on the use of CRISPR/Cas9 genome engineering for sequence-specific genome editing in B. subtilis are provided.

Gradual enhancement of ethyl acetate production through promoter engineering in chinese liquor yeast strains.

As content and proportion of ethyl acetate is critical to the flavor and quality of beverages, the concise regulation of the ethyl acetate metabolism is a major issue in beverage fermentations. In this study, for ethyl acetate yield regulation, we finely modulated the expression of ATF1 through precise and seamless insertion of serially truncated PGK1 promoter from the 3' end by 100bp steps in the Chinese liquor yeast, CLy12a. The three engineered promoters carrying 100-, 200-, and 300-bp truncations exhibited reduced promoter strength but unaffected growth. These three promoters were integrated into the CLy12a strain, generating strains CLy12a-P-100, CLy12a-P-200, and CLy12a-P-300, respectively. The transcription levels of CLy12a-P-100, CLy12a-P-200, and CLy12a-P-300 were 20%, 17%, and 10% of that of CLy12a-P, respectively. The AATase (alcohol acetyl transferases, encoded by the ATF1 gene) activity of three engineered strains were 36%, 56%, and 62% of that of CLy12a-P. In the liquid fermentation of corn hydrolysate at 30°C, the concentration of ethyl acetate in CLy12a-P-100, CLy12a-P-200, and CLy12a-P-300 were reduced by 28%, 30%, and 42%, respectively, compared to CLy12a-P. These results verifying that the ethyl acetate yield could be gradually enhanced by finely modulating the expression of ATF1. The engineered strain CLy12a-P-200 produced the ethyl acetate concentration with the best sensorial quality compared to the other engineered yeast strains. The method proposed in this work supplies a practical proposal for breeding Chinese liquor yeast strains with finely modulated ethyl acetate yield. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:328-336, 2018.