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1.
Appl Microbiol Biotechnol ; 108(1): 190, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38305911

RESUMO

Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique to detect point mutation and screen saturation mutation library. In this study, mismatch amplification mutation assay (MAMA) PCR was used to detect point mutation and screen saturation mutation library. In order to fine-tune the expression of odhA encoding 2-oxoglutarate dehydrogenase E1 component, a saturating mutant library of the RBS of odhA was created in Corynebacterium glutamicum P12 based on the CRISPR-Cas2a genome editing system, which increased the L-proline production by 81.3%. MAMA PCR was used to filter out 42% of the non-mutant transformants in the mutant library, which effectively reduced the workload of the subsequent fermentation test and the number of sequenced samples. The rapid and sensitive MAMA-PCR method established in this study provides a general strategy for detecting point mutations and improving the efficiency of mutation library screening. KEY POINTS: • MAMA PCR was optimized and developed to detect point mutation. • MAMA PCR greatly improves the screening efficiency of point mutation. • Attenuation of odhA expression in P12 effectively improves proline production.


Assuntos
Corynebacterium glutamicum , Mutação Puntual , Mutação , Sequência de Bases , Corynebacterium glutamicum/genética , Reação em Cadeia da Polimerase/métodos
2.
Int Microbiol ; 26(1): 11-24, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35925494

RESUMO

The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMICg::ISm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDHCg) by NADP-GapDHCa from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ significantly between these strains (14.1 ± 1.8 g/L vs 16.2 ± 1.1 g/L). Enhancing the carbon flux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icdCg were introduced into C. glutamicum with NADP-GapDHCa and pEC-ABNCE increased L-leucine yield (54.3 ± 2.9 g/L) and improved cell growth (OD562 = 83.4 ± 7.5) in fed-batch fermentation because the resultant strain C. glutamicum XL-1 ΔMICg::ISm GCg::GCa Pzwf-D1 Picd-D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the first report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.


Assuntos
Clostridium acetobutylicum , Corynebacterium glutamicum , NADP/genética , NADP/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Leucina/genética , Leucina/metabolismo , Clostridium acetobutylicum/metabolismo , Fermentação
3.
Appl Microbiol Biotechnol ; 107(11): 3593-3603, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37097502

RESUMO

L-arginine (L-Arg) is a semi-essential amino acid with many important physiological functions. However, achieving efficient manufacture of L-Arg on an industrial scale using Escherichia coli (E. coli) remains a major challenge. In previous studies, we constructed a strain of E. coli A7, which had good L-Arg production capacity. In this study, E. coli A7 was further modified, and E. coli A21 with more efficient L-Arg production capacity was obtained. Firstly, we reduced the acetate accumulation of strain A7 by weakening the poxB gene and overexpressing acs gene. Secondly, we improved the L-Arg transport efficiency of strains by overexpressing the lysE gene from Corynebacterium glutamicum (C. glutamicum). Finally, we enhanced the supplies of precursors for the synthesis of L-Arg and optimized the supplies of cofactor NADPH and energy ATP in strain. After fermentation in a 5-L bioreactor, the L-Arg titer of strain A21 was found to be 89.7 g/L. The productivity was 1.495 g/(L·h) and the glucose yield was 0.377 g/g. Our study further narrowed the titer gap between E. coli and C. glutamicum in the synthesis of L-Arg. In all recent studies on the L-Arg production by E. coli, this was the highest titer recorded. In conclusion, our study further promotes the efficient mass synthesis of L-Arg by E. coli. KEY POINTS: • The acetate accumulation of starting strain A7 was decreased. • Overexpression of gene lysE of C. glutamicum enhanced L-Arg transport in strain A10. • Enhance the supplies of precursors for the synthesis of L-Arg and optimize the supplies of cofactor NADPH and energy ATP. Finally, Strain A21 was detected to have an L-Arg titer of 89.7 g/L in a 5-L bioreactor.


Assuntos
Corynebacterium glutamicum , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Arginina/metabolismo , NADP/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Trifosfato de Adenosina/metabolismo , Engenharia Metabólica , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo
4.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675279

RESUMO

α-Farnesene, an acyclic volatile sesquiterpene, plays important roles in aircraft fuel, food flavoring, agriculture, pharmaceutical and chemical industries. Here, by re-creating the NADPH and ATP biosynthetic pathways in Pichia pastoris, we increased the production of α-farnesene. First, the native oxiPPP was recreated by overexpressing its essential enzymes or by inactivating glucose-6-phosphate isomerase (PGI). This revealed that the combined over-expression of ZWF1 and SOL3 increases α-farnesene production by improving NADPH supply, whereas inactivating PGI did not do so because it caused a reduction in cell growth. The next step was to introduce heterologous cPOS5 at various expression levels into P. pastoris. It was discovered that a low intensity expression of cPOS5 aided in the production of α-farnesene. Finally, ATP was increased by the overexpression of APRT and inactivation of GPD1. The resultant strain P. pastoris X33-38 produced 3.09 ± 0.37 g/L of α-farnesene in shake flask fermentation, which was 41.7% higher than that of the parent strain. These findings open a new avenue for the development of an industrial-strength α-farnesene producer by rationally modifying the NADPH and ATP regeneration pathways in P. pastoris.


Assuntos
Pichia , Sesquiterpenos , NADP/metabolismo , Pichia/genética , Pichia/metabolismo , Sesquiterpenos/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Engenharia Metabólica
5.
Int J Mol Sci ; 24(14)2023 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-37511626

RESUMO

L-tryptophan (L-trp), produced through bio-manufacturing, is widely used in the pharmaceutical and food industries. Based on the previously developed L-trp-producing strain, this study significantly improved the titer and yield of L-trp, through metabolic engineering of the shikimate pathway and the L-tryptophan branch. First, the rate-limiting steps in the shikimate pathway were investigated and deciphered, revealing that the combined overexpression of the genes aroE and aroD increased L-trp production. Then, L-trp synthesis was further enhanced at the shaking flask level by improving the intracellular availability of L-glutamine (L-gln) and L-serine (L-ser). In addition, the transport system and the competing pathway of L-trp were also modified, indicating that elimination of the gene TnaB contributed to the extracellular accumulation of L-trp. Through optimizing formulas, the robustness and production efficiency of engineered strains were enhanced at the level of the 30 L fermenter. After 42 h of fed-batch fermentation, the resultant strain produced 53.65 g/L of L-trp, with a yield of 0.238 g/g glucose. In this study, the high-efficiency L-trp-producing strains were created in order to establish a basis for further development of more strains for the production of other highly valuable aromatic compounds or their derivatives.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Triptofano/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Fermentação
6.
World J Microbiol Biotechnol ; 40(1): 17, 2023 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-37981595

RESUMO

Microorganisms play an important role in regulating flavor compounds in rice wine, whereas we often don't understand how did they affect flavor compounds. Here, the relations between flavor compounds and microbial community ecological succession were investigated by monitoring flavor compounds and microbial community throughout the fermentation stage of rice wine. The composition of microbial community showed a dynamic change, but 13 dominant bacterial genera and 4 dominant fungal genera were detected throughout the fermentation stages. Saccharomyces presented a strong negative correlation with fungi genera but had positive associations with bacteria genera. Similarly, flavor compounds in rice wine were also showed the dynamic change, and 112 volatile compounds and 17 free amino acids were identified in the whole stages. The alcohol-ester ratio was decreased in the LTF stage, indicating that low temperature boosts ester formation. The potential correlation between flavor compounds and microbial community indicated that Delftia, Chryseobacterium, Rhizopus and Wickerhamomyces were the core functional microorganisms in rice wine. These findings clarified the correlation between changes in flavor compounds and in microbial community in the liquid fermentation of rice wine, and these results have some reference value for the quality improvement and technological optimization in liquid fermentation of rice wine.


Assuntos
Microbiota , Vinho , Fermentação , Suplementos Nutricionais , Ésteres
7.
Appl Microbiol Biotechnol ; 106(17): 5603-5613, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35931894

RESUMO

As an important semi-essential amino acid, L-arginine (L-Arg) has important application prospects in medicine and health care. However, it remains a challenge to efficiently produce L-Arg by Escherichia coli (E. coli). In the present study, we obtained an E. coli A1 with L-Arg accumulation ability, and carried out a series of metabolic engineering on it, and finally obtained an E. coli strain A7 with high L-Arg production ability. First, genome analysis of strain A1 was performed to explore the related genes affecting L-Arg accumulation. We found that gene speC and gene speF played an important role in the accumulation of L-Arg. Second, we used two strategies to solve the feedback inhibition of the L-Arg pathway in E. coli. One was the combination of a mutation of the gene argA and the deletion of the gene argR, and the other was the combination of a heterologous insertion of the gene argJ and the deletion of the gene argR. The combination of exogenous argJ gene insertion and argR gene deletion achieved higher titer accumulation with less impact on strain growth. Finally, we inserted the gene cluster argCJBDF of Corynebacterium glutamicum (C. glutamicum) to enhance the metabolic flux of the L-Arg pathway in E. coli. The final strain obtained 70.1 g/L L-Arg in a 5-L bioreactor, with a yield of 0.326 g/g glucose and a productivity of 1.17 g/(L· h). This was the highest level of L-Arg production by E. coli ever reported. Collectively, our findings provided valuable insights into the possibility of the industrial production of L-Arg by E. coli. KEY POINTS: • Genetic background of E. coli A1 genome analysis. • Heterologous argJ substitution of argA mutation promoted excessive accumulation of L-Arg in E. coli A1. • The overexpression of L-Arg synthesis gene cluster argCJBDF of Corynebacterium glutamicum (C. glutamate) promoted the accumulation of L-Arg, and 70.1 g/L L-Arg was finally obtained in fed-batch fermentation.


Assuntos
Corynebacterium glutamicum , Engenharia Metabólica , Arginina , Escherichia coli , Fermentação
8.
Appl Microbiol Biotechnol ; 106(1): 145-160, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34870736

RESUMO

This article focuses on engineering Corynebacterium glutamicum to produce L-lysine efficiently from starch using combined method of "classical breeding" and "genome breeding." Firstly, a thermo-tolerable L-lysine-producing C. glutamicum strain KT45-6 was obtained after multi-round of acclimatization at high temperature. Then, amylolytic enzymes were introduced into strain KT45-6, and the resultant strains could use starch for cell growth and L-lysine production except the strain with expression of isoamylase. In addition, co-expression of amylolytic enzymes showed a good performance in starch degradation, cell growth and L-lysine production, especially co-expression of α-amylase (AA) and glucoamylase (GA). Moreover, L-lysine yield was increased by introducing AA-GA fusion protein (i.e., strain KT45-6S-5), and finally reached to 23.9 ± 2.3 g/L in CgXIIIPM-medium. It is the first report of an engineered L-lysine-producing strain with maximum starch utilization that may be used as workhorse for producing amino acid using starch as the main feedstock. KEY POINTS: • Thermo-tolerable C. glutamicum was obtained by temperature-induced adaptive evolution. • The fusion order between AA and GA affects the utilization efficiency of starch. • C. glutamicum with starch utilization was constructed by optimizing amylases expression.


Assuntos
Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Fermentação , Lisina/metabolismo , Engenharia Metabólica , Amido/metabolismo , Zea mays/metabolismo
9.
World J Microbiol Biotechnol ; 38(2): 22, 2022 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-34989926

RESUMO

As an important raw material for pharmaceutical, food and feed industry, highly efficient production of L-tryptophan by Escherichia coli has attracted a considerable attention. However, there are complicated and multiple layers of regulation networks in L-tryptophan biosynthetic pathway and thus have difficulty to rewrite the biosynthetic pathway for producing L-tryptophan with high efficiency in E. coli. This review summarizes the biosynthetic pathway of L-tryptophan and highlights the main regulatory mechanisms in E. coli. In addition, we discussed the latest metabolic engineering strategies achieved in E. coli to reconstruct the L-tryptophan biosynthetic pathway. Moreover, we also review a few strategies that can be used in E. coli to improve robustness and streamline of L-tryptophan high-producing strains. Lastly, we also propose the potential strategies to further increase L-tryptophan production by systematic metabolic engineering and synthetic biology techniques.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Triptofano/biossíntese , Triptofano/genética , Aminoácidos Aromáticos/biossíntese , Aminoácidos Aromáticos/genética , Vias Biossintéticas/genética , Biotecnologia/métodos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética
10.
Amino Acids ; 53(9): 1301-1312, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34401958

RESUMO

L-valine is an essential branched-chain amino acid that cannot be synthesized by the human body and has a wide range of applications in food, medicine and feed. Market demand has stimulated people's interest in the industrial production of L-valine. At present, the mutagenized or engineered Corynebacterium glutamicum is an effective microbial cell factory for producing L-valine. Because the biosynthetic pathway and metabolic network of L-valine are intricate and strictly regulated by a variety of key enzymes and genes, highly targeted metabolic engineering can no longer meet the demand for efficient biosynthesis of L-valine. In recent years, the development of omics technology has promoted the upgrading of traditional metabolic engineering to systematic metabolic engineering. This whole-cell-scale transformation strategy has become a productive method for developing L-valine producing strains. This review provides an overview of the biosynthesis and regulation mechanism of L-valine, and summarizes the current metabolic engineering techniques and strategies for constructing L-valine high-producing strains. Finally, the opinion of constructing a cell factory for efficiently biosynthesizing L-valine was proposed.


Assuntos
Vias Biossintéticas , Corynebacterium glutamicum/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Valina/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Fermentação , Humanos
11.
Appl Microbiol Biotechnol ; 105(24): 9153-9166, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34837493

RESUMO

To directly produce ß-alanine from glucose by microbial fermentation, a recombinant Corynebacterium glutamicum strain with high efficiency of ß-alanine production was constructed in this study. To do this, the biosynthetic pathway of ß-alanine in an L-lysine-producing strain XQ-5 was modified by enhancing carbon flux in biosynthetic pathway and limiting carbon flux in competitive pathway. This study showed that replacement of L-aspartate kinase (AK) with wild-type AK and disruption of lactate dehydrogenase and alanine/valine aminotransferases increase ß-alanine production because of decreasing the by-products accumulation. Moreover, L-aspartate-α-decarboxylase (ADC) from Bacillus subtilis was designed as the best enzyme for increasing ß-alanine production, and its variant (BsADCE56S/I88M) showed the highest activity for catalyzing L-aspartate to generate ß-alanine. To further increase ß-alanine production, expression level of BsADCE56S/I88M was controlled by optimizing promoter and RBS, indicating that Pgro plus ThirRBS is the best combination for BsADCE56S/I88M expression and ß-alanine production. The resultant strain XQ-5.5 produced 30.7 ± 2.3 g/L of ß-alanine with a low accumulation of lactate (from 5.2 ± 0.14 to 0.2 ± 0.09 g/L) and L-alanine (from 7.6 ± 0.22 to 3.8 ± 0. 32 g/L) in shake-flask fermentation and produced 56.5 ± 3.2 g/L of ß-alanine with a productivity of 0.79 g/(L·h) and the glucose conversion efficiency (α) of 39.5% in feed-batch fermentation. This is the first report of genetically modifying the biosynthetic pathway of ß-alanine that improves the efficiency of ß-alanine production in an L-lysine-producing strain, and these results give us a new insight for constructing the other valuable biochemical. KEY POINTS: • Optimization and overexpression of the key enzyme BsADC increased the accumulation of ß-alanine. • The AK was replaced with wild-type AK to increase the conversion of aspartic acid to ß-alanine. • A 56.5-g/L ß-alanine production in fed-batch fermentation was achieved.


Assuntos
Corynebacterium glutamicum , Vias Biossintéticas/genética , Corynebacterium glutamicum/genética , Glucose , Engenharia Metabólica , beta-Alanina
12.
Ecotoxicol Environ Saf ; 220: 112328, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34015636

RESUMO

Agricultural runoff is an important antibiotic resistance genes (ARGs) dissemination pathway from farmlands to water environment, however few studies have focused on the influence of agricultural land-use change on the pattern of ARGs in runoff and assess the health risk to public. Lake Tai Basin which experiences agricultural land-use change was selected to elucidate this concern. Our findings revealed that the pattern of ARGs was more diverse and the gene abundance was higher in orchard runoffs by comparison with conventional cropland runoffs. Co-occurrence network analysis between mobile genetic elements and ARGs demonstrated that after agricultural land-use change, ARG dissemination via runoffs became more threatened. In addition, this study illustrated the correlations between the antibiotic resistome and microbiome in runoffs, finding that non-dominant microbial taxa were the limiting factor which determined the pattern of ARGs in surface runoffs. In summary, the pattern and dissemination risk of ARGs in the surface runoff after agricultural land-use change in Lake Tai Basin were clarified via this study.


Assuntos
Agricultura , Antibacterianos/farmacologia , Bactérias/genética , Microbiologia do Solo , Poluentes do Solo/análise , China , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos
13.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445771

RESUMO

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.


Assuntos
Corynebacterium glutamicum/metabolismo , Ácido Diaminopimélico/metabolismo , Lisina/metabolismo , Transdução de Sinais/fisiologia , Aciltransferases/metabolismo , Compostos de Amônio/metabolismo , Oxirredutases/metabolismo
14.
Macromol Rapid Commun ; 41(15): e2000260, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32648310

RESUMO

Polymerization-induced self-assembly has been demonstrated to be a powerful strategy for fabricating polymeric nanoparticles in the last two decades. However, the stringent requirements for the monomers greatly limit the chemical versatility of PISA-based functional nanoparticles and expanding the monomer family of PISA is still highly desirable. Herein, a camptothecin analogue (CPTM) is first used as the monomer in PISA. Prodrug nanoparticles with reduction-responsive camptothecin release behavior are fabricated at 10% solid concentration (100 mg g-1 ). Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) and poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) are used as the macro RAFT agents to comediate the RAFT dispersion polymerization of CPTM in ethanol to produce the PHPMA/PDEAEMA-stabilized nanoparticles. The PDEAEMA chains become hydrophobic and are in the collapsed state at physiological pH values. In contrast, in the vicinity of an acidic tumor, the tertiary amine groups of PDEAEMA chains are rapidly protonated, leading to fast hydrophobic-hydrophilic transitions and charge reversal. Such fast charge-reversal results in enhanced cancer cell internalization of the prodrug nanoparticles, thus achieving superior anticancer efficacy.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Pró-Fármacos/química , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Etanol/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Metacrilatos/química , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Nylons/química , Polimerização , Polímeros/química , Ácidos Polimetacrílicos/química , Água/química
15.
J Ind Microbiol Biotechnol ; 47(6-7): 485-495, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32535763

RESUMO

L-Leucine is an essential amino acid that has wide and expanding applications in the industry. It is currently fast-growing market demand that provides a powerful impetus to further increase its bioconversion productivity and production stability. In this study, we rationally engineered the metabolic flux from pyruvate to L-leucine synthesis in Corynebacterium glutamicum to enhance both pyruvate availability and L-leucine synthesis. First, the pyc (encoding pyruvate carboxylase) and avtA (encoding alanine-valine aminotransferase) genes were deleted to weaken the metabolic flux of the tricarboxylic acid cycle and reduce the competitive consumption of pyruvate. Next, the transcriptional level of the alaT gene (encoding alanine aminotransferase) was down regulated by inserting a terminator to balance L-leucine production and cell growth. Subsequently, the genes involved in L-leucine biosynthesis were overexpressed by replacing the native promoters PleuA and PilvBNC of the leuA gene and ilvBNC operon, respectively, with the promoter Ptuf of eftu (encoding elongation factor Tu) and using a shuttle expression vector. The resulting strain WL-14 produced 28.47 ± 0.36 g/L L-leucine in shake flask fermentation.


Assuntos
Carbono/metabolismo , Corynebacterium glutamicum/metabolismo , Leucina/biossíntese , Alanina/biossíntese , Ciclo do Ácido Cítrico , Corynebacterium glutamicum/genética , Fermentação , Microbiologia Industrial , Engenharia Metabólica , Plasmídeos/metabolismo , Ácido Pirúvico/metabolismo , Transaminases/metabolismo , Valina/biossíntese
16.
Zhongguo Zhong Yao Za Zhi ; 45(15): 3603-3607, 2020 Aug.
Artigo em Zh | MEDLINE | ID: mdl-32893549

RESUMO

Osteoporosis fracture with high disability and mortality is a difficult problem that seriously affects the life quality of individuals. At present, there is still a lack of anti-osteoporosis drugs with clear target and significant efficacy in the clinical practice. Rehmanniae Radix and its prescriptions have significant clinical effects. In this regard, more and more studies have reported the effects and mechanisms of Rehmanniae Radix and its active components, and the certain research outputs have been achieved. In this article, the PubMed, Web of science, China National Knowledge Infrastructure, and Wanfang database were searched to collect and organize the latest research progress of Rehmanniae Radix treatment of osteoporosis in the recent 10 years. We summarized the research dynamics as well as the function indexes and mechanisms of the raw and processed Rehmanniae Radix, active ingredients such as catalpol, aucubin, acteoside and Rehmanniae Radix polysaccharide, and their formulating prescriptions, and then excavated the potential active ingredients, targets and signaling pathways, including the effect on bone marrow mesenchymal stem cells, promoting the osteoblast proliferation and promoting osteogenesis differentiation(increasing alkaline phosphatase, typeⅠ collagen, osteoprotegerin, and osteocalcin and promoting calcium deposits), increasing the bone density, inhibiting the osteoclast quantity and differentiation, promoting the osteoclast apoptosis, and reducing tartrate resistant acid phosphatase and bone resorption pit area to provide the reference and develop new ideas for developing Rehmanniae Radix prescriptions for treatment of osteoporosis and exploring its mechanism.


Assuntos
Medicamentos de Ervas Chinesas , Osteoporose , Rehmannia , China , Humanos , Osteogênese
17.
Zhongguo Dang Dai Er Ke Za Zhi ; 22(6): 561-566, 2020 Jun.
Artigo em Zh | MEDLINE | ID: mdl-32571452

RESUMO

OBJECTIVE: To investigate the current status of antibiotic use for very and extremely low birth weight (VLBW/ELBW) infants in neonatal intensive care units (NICUs) of Hunan Province. METHODS: The use of antibiotics was investigated in multiple level 3 NICUs of Hunan Province for VLBW and ELBW infants born between January, 2017 and December, 2017. RESULTS: The clinical data of 1 442 VLBW/ELBW infants were collected from 24 NICUs in 2017. The median antibiotic use duration was 17 days (range: 0-86 days), accounting for 53.0% of the total length of hospital stay. The highest duration of antibiotic use was up to 91.4% of the total length of hospital stay, with the lowest at 14.6%. In 16 out of 24 NICUs, the antibiotic use duration was accounted for more than 50.0% of the hospitalization days. There were 113 cases with positive bacterial culture grown in blood or cerebrospinal fluid, making the positive rate of overall bacterial culture as 7.84%. The positive rate of bacterial culture in different NICUs was significantly different from 0% to 14.9%. The common isolated bacterial pathogens Klebsiella pneumoniae was 29 cases (25.7%); Escherichia coli 12 cases (10.6%); Staphylococcus aureus 3 cases (2.7%). The most commonly used antibiotics were third-generation of cephalosporins, accounting for 41.00% of the total antibiotics, followed by penicillins, accounting for 32.10%, and followed by carbapenems, accounting for 13.15%. The proportion of antibiotic use time was negatively correlated with birth weight Z-score and the change in weight Z-score between birth and hospital discharge (rs=-0.095, -0.151 respectively, P<0.01), positively correlated with death/withdrawal of care (rs=0.196, P<0.01). CONCLUSIONS: Antibiotics used for VLBW/ELBW infants in NICUs of Hunan Province are obviously prolonged in many NICUs. The proportion of routine use of third-generation of cephalosporins and carbapenems antibiotics is high among the NICUs.


Assuntos
Recém-Nascido de Peso Extremamente Baixo ao Nascer , Antibacterianos , Peso ao Nascer , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Inquéritos e Questionários
18.
Crit Rev Biotechnol ; 39(5): 633-647, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31055970

RESUMO

l-Leucine, as an essential branched-chain amino acid for humans and animals, has recently been attracting much attention because of its potential for a fast-growing market demand. The applicability ranges from flavor enhancers, animal feed additives and ingredients in cosmetic to specialty nutrients in pharmaceutical and medical fields. Microbial fermentation is the major method for producing l-leucine by using Escherichia coli and Corynebacterium glutamicum as host bacteria. This review gives an overview of the metabolic pathway of l-leucine (i.e. production, import and export systems) and highlights the main regulatory mechanisms of operons in E. coli and C. glutamicum l-leucine biosynthesis. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating l-leucine producing strains. Finally, future perspectives to construct industrially advantageous strains are considered with respect to recent advances in biology.


Assuntos
Corynebacterium glutamicum/metabolismo , Escherichia coli/metabolismo , Leucina/biossíntese , Corynebacterium glutamicum/genética , Escherichia coli/genética , Leucina/genética , Engenharia Metabólica , Óperon
19.
Appl Opt ; 58(7): 1691-1698, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30874200

RESUMO

In view of the optical detection requirements of wide-area and continuous surveillance of air targets, the detection ability of an infrared imaging system in geostationary orbit for aircraft plumes is studied. The point spread model of the subpixel imaging of the full chain, including the aircraft plume, the sea surface, the environmental atmosphere, the optical system, and the imaging detector is established. The detection ability of the typical imaging system in geostationary orbit is analyzed from the signal-to-noise ratio (SNR) and the detection range in combination with the effect of the point spread function (PSF) of the optical system. Meanwhile, the optimal coupling condition of the PSF to the spatial resolution is discussed. The imaging characteristics of the aircraft target on the focal plane of the infrared imaging system under different spatial sampling rates are simulated and verified. Research shows that the SNR of the system first increases, and then decreases gradually with an increase of the spatial sampling rate. The detectable range covered by the pixel footprint decreases as the detector size increases. When the detector size is 15, 20, and 30 µm, the target can be detected with a spatial resolution of 200-700, 300-700, and 400-600 m, respectively.

20.
Appl Opt ; 58(18): 4835-4845, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31503798

RESUMO

The imaging quality of an airborne infrared (IR) system is limited by the angular disturbance of the airborne platform. Based on the full-chain (IR scene-atmosphere-optical system-detector-airborne platform) signal transmission process, this study focused on the low-frequency sinusoidal angular disturbance features of the airborne platform and accurately calculated the point spread function caused by the angular disturbance and the IR imaging features when the IR system's different locations were dynamically simulated in a three-dimensional scene. First, the degradation mechanism of the IR imaging features resulting from the angular disturbance was analyzed from the viewpoint of scene radiation signal transmission and detector sampling. Then, the dynamic simulation in the three-dimensional scene resulting from the angular disturbance was realized by considering the geometric transformation of the spatial imaging, scale registration of the spatial sampling, radiation coupling, and angular disturbance caused by the airborne platform. Finally, the distances detected under different disturbance conditions were predicted using the established model. The obtained results provide data supporting the demonstration, verification, and optimization of the IR imaging system's design scheme.

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