Effect of microwave alone and microwave-assisted modification on the physicochemical properties of starch and its application in food.

Native starch has poor stability and usually requires modification to expand its industrial application range. Commonly used methods are physical, chemical, enzymatic and compound modification. Microwave radiation, as a kind of physical method, is promising due to its uniform energy radiation, greenness, safety, non-toxicity. It can meet the demand of consumers for safe food. Microwave-assisted modification with other methods can directly or indirectly affect the structure of starch granules to obtain modified starch with high degree of substitution and low viscosity, and the modification efficiency is greatly improved. This paper reviews the effect of microwave radiation on the physicochemical properties of starch, such as granule morphology, crystallization characteristics, and gelatinization characteristics, as well as the application of microwave radiation in starch modification and starch food processing. It provides theoretical references and suggestions for the research of microwave heating modified starch and the deep processing of starchy foods.

Enhancing the capability of Klebsiella pneumoniae to produce 1, 3-propanediol by overexpression and regulation through CRISPR-dCas9.

Klebsiella pneumoniae is a common strain of bacterial fermentation to produce 1, 3-propanediol (1, 3-PDO). In general, the production of 1, 3-PDO by wild-type K. pneumoniae is relatively low. Therefore, a new gene manipulation of K. pneumoniae was developed to improve the production of 1, 3-PDO by overexpressing in the reduction pathway and attenuating the by-products in the oxidation pathway. Firstly, dhaB and/or dhaT were overexpressed in the reduction pathway. Considering the cost of IPTG, the constitutive promoter P32 was selected to express the key gene. By comparing K.P. pET28a-P32-dhaT with the original strain, the production of 1, 3-PDO was increased by 19.7%, from 12.97 to 15.53 g l-1 (in a 250 ml shaker flask). Secondly, three lldD and budC regulatory sites were selected in the by-product pathway, respectively, using the CRISPR-dCas9 system, and the optimal regulatory sites were selected following the 1, 3-PDO production. As a result, the 1, 3-PDO production by K.P. L1-pRH2521 and K.P. B3-pRH2521 reached up to 19.16 and 18.74 g l-1 , which was increased by 47.7% and 44.5% respectively. Overexpressing dhaT and inhibiting expression of lldD and budC were combined to further enhance the ability of K. pneumoniae to produce 1, 3-PDO. The 1, 3-PDO production by K.P. L1-B3-PRH2521-P32-dhaT reached 57.85 g l-1 in a 7.5 l fermentation tank (with Na+ neutralizer), which is higher than that of the original strain. This is the first time that the 1, 3-PDO production was improved in K. pneumoniae by overexpressing the key gene and attenuating by-product synthesis in the CRISPR-dCas9 system. This study reports an efficient approach to regulate the expression of genes in K. pneumoniae to increase the 1, 3-PDO production, and such a strategy may be useful to modify other strains to produce valuable chemicals.

Improvement and Metabolomics-Based Analysis of d-Lactic Acid Production from Agro-Industrial Wastes by Lactobacillus delbrueckii Submitted to Adaptive Laboratory Evolution.

To decrease d-lactic acid production cost, sugarcane molasses and soybean meal, low-cost agro-industrial wastes, were selected as feedstock. First, sugarcane molasses was used directly by Lactobacillus delbrueckii S-NL31, and the nutrients were released from soybean meal by protease hydrolysis. Subsequently, to ensure intensive substrate utilization and enhanced d-lactic acid production from sugarcane molasses and soybean meal, adaptation of L. delbrueckii S-NL31 to substrates was performed through adaptive laboratory evolution. After two-phase adaptive laboratory evolution, the evolved strain L. delbrueckii S-NL31-CM3-SBM with improved cell growth and d-lactic acid production on sugarcane molasses and soybean meal was obtained. To decipher the potential reasons for improved fermentation performance, a metabolomics-based approach was developed to profile the differences of intracellular metabolism between initial and evolved strain. The in-depth analysis elucidated how the key factors exerted influence on d-lactic acid biosynthesis. The results revealed that the enhancement of glycolysis pathway and cofactor supply was directly associated with increased lactic acid production, and the reinforcement of pentose phosphate pathway, amino acid metabolism, and oleic acid uptake improved cell survival and growth. These might be the main reasons for significantly improved d-lactic acid production by adaptive laboratory evolution. Finally, fed-batch simultaneous enzymatic hydrolysis of soybean meal and fermentation process by evolved strain resulted in d-lactic acid levels of 112.3 g/L, with an average production efficiency of 2.4 g/(L × h), a yield of 0.98 g/g sugar, and optical purity of 99.6%. The results show the applicability of d-lactic acid production in L. delbrueckii fed on agro-industrial wastes through adaptive laboratory evolution.

Negative regulation of bleomycins biosynthesis by ArsR/SmtB family repressor BlmR in Streptomyces verticillus.

Bleomycin, a broad-spectrum antibiotic, has been widely used for various tumor treatments. However, its poor fermentation yield is not satisfactory for industrial production. Here, the ArsR/SmtB family regulator BlmR was characterized as a repressor of bleomycin production. As an autoregulator, BlmR was found to bind to a 12-2-12 imperfect palindrome sequence in its own promoter, and deletion of blmR led to a 34% increase of bleomycin B2 production compared with the wild-type strain. Using reverse transcription and quantitative PCR (RT-qPCR), blmT, which encoded a putative transporter, was identified as the target gene regulated by BlmR. Therefore, high-production strain was constructed by blmT overexpression in a blmR deletion strain, and the bleomycin B2 titer reached to 80 mg/L, which was 1.9-fold higher than the wild-type strain. Moreover, electrophoretic mobility shift assay (EMSA) showed neither metal-binding motifs nor redox switches in BlmR. In order to elucidate the regulatory mechanism, a model of BlmR was constructed by homology modeling and protein-protein docking. The BlmR-DNA complex was generated by protein-DNA docking with the assistance of site-directed mutagenesis and molecular dynamic (MD) simulation, which directly revealed several key amino acid residues needed for the maintenance and stabilization of the interface between BlmR and target DNA. The interface information could provide the configuration reference and seek the potential effectors that could interact with BlmR, thereby extending the regulation role of ArsR/SmtB family members on the improvement of antibiotic production.

Enhancing the production of tacrolimus by engineering target genes identified in important primary and secondary metabolic pathways and feeding exogenous precursors.

Tacrolimus has been widely used as a powerful novel immunosuppressant. The objective of this study was to improve the production of tacrolimus by engineering the target genes of important primary and secondary metabolic pathways and feeding exogenous precursors. Based on the metabonomics analysis, the shikimic acid pathway is an important primary metabolic pathway for the producing tacrolimus. Combined overexpression of shikimate kinase and dehydroquinic acid synthetase genes led to a 33.1% enhancement of tacrolimus production compared to parent strain. To predict the most efficient targets in secondary metabolic pathways for improving the production of tacrolimus, a genome-scale dynamic metabolic network model was used. A knockout of the D-lactate dehydrogenase gene, combined with the overexpression of tryptophane synthase and aspartate 1-decarboxylase genes, led to a 29.8% enhancement of tacrolimus production compared to the parent strain. Finally, we investigated the impact of the genetic manipulations on transcription levels, cell growth, cell morphology and production of tacrolimus by qRT-PCR and scanning electron microscopy to reveal the relationship between the growth of strains, the effects of engineering and fermentation. As the efficient synthesis of tacrolimus requires a rich supply of external substrates, the efficiency of the metabolic pathways that convert these substances is extremely important. The combined addition of three external substrates such as shikimic acid, alanine and the n-dodecane increased tacrolimus production by 49.5%. The insights obtained in this study will help further elucidate the mechanisms by which the identified target genes promote the activity of important primary and secondary metabolic pathways for tacrolimus biosynthesis and provide a new feeding strategy to improve tacrolimus production.

Metabolic engineering of Escherichia coli for 1,3-propanediol biosynthesis from glycerol.

In this study, the engineered E. coli was constructed for efficient transformation of glycerol to 1,3-propanediol (1,3-PDO). To regenerate NADPH, the key bottleneck in 1,3-PDO production, heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDN, encoded by gapN) pathway was introduced, and the gapN expression level was fine-tuned with specific 5'-untranslated regions (5'-UTR) to balance the carbon flux distribution between the metabolic pathways of NADPH regeneration and 1,3-PDO biosynthesis. Additionally, glucose was added to the medium to promote glycerol utilization and cell growth. To elevate the utilization of glycerol in the presence of glucose, E. coli JA11 was constructed through destroying PEP-dependent glucose transport system while strengthening the ATP-dependent transport system. Subsequent optimization of nitrogen sources further improved 1,3-PDO production. Finally, under the optimal fermentation condition, E. coli JA11 produced 13.47 g/L 1,3-PDO, with a yield of 0.64 mol/mol, increased by 325% and 100% compared with the original engineered E. coli JA03, respectively.

Identification and metabolomic analysis of chemical elicitors for tacrolimus accumulation in Streptomyces tsukubaensis.

Tacrolimus is a widely used immunosuppressive agent in the treatment of various clinical diseases. However, the low fermentation yield seriously limits its further application. To stimulate tacrolimus synthesis, nine chemical elicitors of five groups were evaluated for their effects on tacrolimus accumulation in S. tsukubaensis. The results showed that sodium butyrate (SB), dimethylsulfoxide (DMSO), and LaCl3 could increase tacrolimus accumulation by more than 30%. Cumulative effects of different chemical elicitors exhibited that the highest tacrolimus yield was improved by 64.7% (303.60 mg/L) in DMSO and La treatment, compared to the control. To decipher possible response mechanism, a weighted correlation network analysis (WGCNA) based on metabolomics was employed and datasets showed 13 distinct metabolic modules and 16 hub metabolites were possibly related to the stimulatory roles of DMSO, La, SB, and their combination treatments. The pathway analysis further exhibited that central carbon metabolism, amino acid metabolism, and fatty acid metabolism showed significant differences in the above chemical elicitor treatments. Thereinto, the carboxylation of propionyl-CoA from isoleucine and methionine degradation was first confirmed to be the main source of methylmalonyl-CoA by RT-PCR analysis in DMSO and La treatment. By further strengthening of the supply of methylmalonyl-CoA precursor in DMSO and La treatment, the final tacrolimus yield could reach to 372.12 mg/L, 2.02-fold higher than the control. To our knowledge, this is the first study to unveil the potential mechanism of different chemical elicitor stresses in S. tsukubaensis based on metabolomics, and the established information provide valuable guidance for further improving tacrolimus production.

Metabolomic and proteomic analysis of D-lactate-producing Lactobacillus delbrueckii under various fermentation conditions.

As an important feedstock monomer for the production of biodegradable stereo-complex poly-lactic acid polymer, D-lactate has attracted much attention. To improve D-lactate production by microorganisms such as Lactobacillus delbrueckii, various fermentation conditions were performed, such as the employment of anaerobic fermentation, the utilization of more suitable neutralizing agents, and exploitation of alternative nitrogen sources. The highest D-lactate titer could reach 133 g/L under the optimally combined fermentation condition, increased by 70.5% compared with the control. To decipher the potential mechanisms of D-lactate overproduction, the time-series response of intracellular metabolism to different fermentation conditions was investigated by GC-MS and LC-MS/MS-based metabolomic analysis. Then the metabolomic datasets were subjected to weighted correlation network analysis (WGCNA), and nine distinct metabolic modules and eight hub metabolites were identified to be specifically associated with D-lactate production. Moreover, a quantitative iTRAQ-LC-MS/MS proteomic approach was employed to further analyze the change of intracellular metabolism under the combined fermentation condition, identifying 97 up-regulated and 42 down-regulated proteins compared with the control. The in-depth analysis elucidated how the key factors exerted influence on D-lactate biosynthesis. The results revealed that glycolysis and pentose phosphate pathways, transport of glucose, amino acids and peptides, amino acid metabolism, peptide hydrolysis, synthesis of nucleotides and proteins, and cell division were all strengthened, while ATP consumption for exporting proton, cell damage, metabolic burden caused by stress response, and bypass of pyruvate were decreased under the combined condition. These might be the main reasons for significantly improved D-lactate production. These findings provide the first omics view of cell growth and D-lactate overproduction in L. delbrueckii, which can be a theoretical basis for further improving the production of D-lactate.

Rational design of a synthetic Entner-Doudoroff pathway for enhancing glucose transformation to isobutanol in Escherichia coli.

Isobutanol as a more desirable biofuel has attracted much attention. In our previous work, an isobutanol-producing strain Escherichia coli LA09 had been obtained by rational redox status improvement under guidance of the genome-scale metabolic model. However, the low transformation from sugar to isobutanol is a limiting factor for isobutanol production by E. coli LA09. In this study, the intracellular metabolic profiles of the isobutanol-producing E. coli LA09 with different initial glucose concentrations were investigated and the metabolic reaction of fructose 6-phosphate to 1, 6-diphosphate fructose in glycolytic pathway was identified as the rate-limiting step of glucose transformation. Thus, redesigned carbon catabolism was implemented by altering flux of sugar metabolism. Here, the heterologous Entner-Doudoroff (ED) pathway from Zymomonas mobilis was constructed, and the adaptation of upper and lower parts of ED pathway was further improved with artificial promoters to alleviate the accumulation of toxic intermediate metabolite 2-keto-3-deoxy-6-phospho-gluconate (KDPG). Finally, the best isobutanol-producing E. coli ED02 with higher glucose transformation and isobutanol production was obtained. In the fermentation of strain E. coli ED02 with 45 g/L initial glucose, the isobutanol titer, yield and average producing rate were, respectively, increased by 56.8, 47.4 and 88.1% to 13.67 g/L, 0.50 C-mol/C-mol and 0.456 g/(L × h) in a shorter time of 30 h, compared with that of the starting strain E. coli LA09.

Combining metabolomics and network analysis to improve tacrolimus production in Streptomyces tsukubaensis using different exogenous feedings.

Tacrolimus is widely used as an immunosuppressant in the treatment of various autoimmune diseases. However, the low fermentation yield of tacrolimus has thus far restricted its industrial applications. To solve this problem, the time-series response mechanisms of the intracellular metabolism that were highly correlated with tacrolimus biosynthesis were investigated using different exogenous feeding strategies in S. tsukubaensis. The metabolomic datasets, which contained 93 metabolites, were subjected to weighted correlation network analysis (WGCNA), and eight distinct metabolic modules and seven hub metabolites were identified to be specifically associated with tacrolimus biosynthesis. The analysis of metabolites within each metabolic module suggested that the pentose phosphate pathway (PPP), shikimate and aspartate pathway might be the main limiting factors in the rapid synthesis phase of tacrolimus accumulation. Subsequently, all possible key-limiting steps in the above metabolic pathways were further screened using a genome-scale metabolic network model (GSMM) of S. tsukubaensis. Based on the prediction results, two newly identified targets (aroC and dapA) were overexpressed experimentally, and both of the engineered strains showed higher tacrolimus production. Moreover, the best strain, HT-aroC/dapA, that was engineered to simultaneously enhanced chorismate and lysine biosynthesis was able to produce 128.19 mg/L tacrolimus, 1.64-fold higher than control (78.26 mg/L). These findings represent a valuable addition to our understanding of tacrolimus accumulation in S. tsukubaensis, and pave the way to further production improvements.

Integrating multi-omics analyses of Nonomuraea dietziae to reveal the role of soybean oil in [(4'-OH)MeLeu]4-CsA overproduction.

BACKGROUND: Nonomuraea dietziae is a promising microorganism to mediate the region-specific monooxygenation reaction of cyclosporine A (CsA). The main product [(4'-OH)MeLeu]4-CsA possesses high anti-HIV/HCV and hair growth-stimulating activities while avoiding the immunosuppressive effect of CsA. However, the low conversion efficiency restricts the clinical application. In this study, the production of [(4'-OH)MeLeu]4-CsA was greatly improved by 55.6% from 182.8 to 284.4 mg/L when supplementing soybean oil into the production medium, which represented the highest production of [(4'-OH)MeLeu]4-CsA so far. RESULTS: To investigate the effect of soybean oil on CsA conversion, some other plant oils (corn oil and peanut oil) and the major hydrolysates of soybean oil were fed into the production medium, respectively. The results demonstrated that the plant oils, rather than the hydrolysates, could significantly improve the [(4'-OH)MeLeu]4-CsA production, suggesting that soybean oil might not play its role in the lipid metabolic pathway. To further unveil the mechanism of [(4'-OH)MeLeu]4-CsA overproduction under the soybean oil condition, a proteomic analysis based on the two-dimensional gel electrophoresis coupled with MALDI TOF/TOF mass spectrometry was implemented. The results showed that central carbon metabolism, genetic information processing and energy metabolism were significantly up-regulated under the soybean oil condition. Moreover, the gas chromatography-mass spectrometry-based metabolomic analysis indicated that soybean oil had a great effect on amino acid metabolism and tricarboxylic acid cycle. In addition, the transcription levels of cytochrome P450 hydroxylase (CYP) genes for CsA conversion were determined by RT-qPCR and the results showed that most of the CYP genes were up-regulated under the soybean oil condition. CONCLUSIONS: These findings indicate that soybean oil could strengthen the primary metabolism and the CYP system to enhance the mycelium growth and the monooxygenation reaction, respectively, and it will be a guidance for the further metabolic engineering of this strain.