RESUMO
As a new CRISPR/Cas-derived genome engineering technology, base editing combines the target specificity of CRISPR/Cas and the catalytic activity of nucleobase deaminase to install point mutations at target loci without generating DSBs, requiring exogenous template, or depending on homologous recombination. Recently, researchers have developed a variety of base editing tools in the important industrial strain Corynebacterium glutamicum, and achieved simultaneous editing of two and three genes. However, the multiplex base editing based on CRISPR/Cas9 is still limited by the complexity of multiple sgRNAs, interference of repeated sequence and difficulty of target loci replacement. In this study, multiplex base editing in C. glutamicum was optimized by the following strategies. Firstly, the multiple sgRNA expression cassettes based on individual promoters/terminators was optimized. The target loci can be introduced and replaced rapidly by using a template plasmid and Golden Gate method, which also avoids the interference of repeated sequence. Although the multiple sgRNAs structure is still complicated, the editing efficiency of this strategy is the highest. Then, the multiple gRNA expression cassettes based on Type Ⅱ CRISPR crRNA arrays and tRNA processing were developed. The two strategies only require one single promoter and terminator, and greatly simplify the structure of the expression cassette. Although the editing efficiency has decreased, both methods are still applicable. Taken together, this study provides a powerful addition to the genome editing toolbox of C. glutamicum and facilitates genetic modification of this strain.
Assuntos
Sistemas CRISPR-Cas/genética , Corynebacterium glutamicum/metabolismo , Edição de Genes , Plasmídeos , /metabolismoRESUMO
An enzyme-displaying yeast as a whole-cell biocatalyst is an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, lipase-displaying Pichia pastoris whole-cell was used as a biocatalyst to synthesize diisooctyl adipate in the non-aqueous system. The maximum productivity of diisooctyl adipate was obtained as 85.0% in a 10 mL reaction system. The yield could be reached as high as 97.8% when the reaction system was scaled up to 200 mL. The purity obtained is 98.2% after vacuum distillation. Thus, the lipase-displaying P. pastoris whole-cell biocatalyst was promising in commercial application for diisooctyl adipate synthesis in non-aqueous phase.
Assuntos
Adipatos , Metabolismo , Microbiologia Industrial , Lipase , Metabolismo , Pichia , MetabolismoRESUMO
Yeast surface display involves that the exogenous protein, which was fused with the yeast outer shell cell wall protein, was genetically anchored on the yeast cell surface. It has been widely used in expression and screening of functional protein. Here, we focused on the construction of lipase-displaying systems and its application in enzymatic biosynthesis, such as fatty acid methyl esters, short-chain flavour esters and sugar esters applications, and so on.
Assuntos
Candida , Genética , Lipase , Genética , Pichia , Genética , Proteínas Recombinantes , Genética , Solventes , Leveduras , GenéticaRESUMO
An enzyme-displaying yeast as a whole-cell biocatalyst seemed an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, we tried to use a recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) to catalyze the synthesis of short chain flavor esters in n-heptane. We studied some major influential factors of esterification reactions, such as carbon chain length of the substrates, alcohol structure, enzyme concentration, substrates concentration, molar ratio of the substrates. The acid conversions were determined by titration and gas chromatography analysis. About ten kinds of esters were synthesized successfully, and the acid conversions of eight esters reached as high as 90% after reaction for 6 h. The result also indicated that ethanol and hexanoic acid were the most suitable substrates for this whole-cell catalyst. Under the optimal reaction conditions (the amount of lipase 20 g/L (306.0 U/g-dry cell), hexanoic acid concentration 0.8 mol/L, the molar ratio of hexanoic acid to ethanol 1:1.1), hexanoic acid conversion reached 97.3% after reaction for 1.5 h. To our knowledge, the CALB-displaying P. pastoris whole-cell biocatalyst showed good tolerance for high substrates concentration and exhibited high reaction rate on esterification of short chain flavor esters among the present enzyme/cell reported. Thus, CALB-displaying P pastoris whole-cell biocatalyst was promising in commercial application for flavor esters synthesis in non-aqueous phase.
Assuntos
Biocatálise , Candida , Enzimas Imobilizadas , Ésteres , Metabolismo , Proteínas Fúngicas , Lipase , Genética , Pichia , Genética , Metabolismo , Proteínas Recombinantes , GenéticaRESUMO
We developed a new enzymatic-catalyzing producing process of glucose laurate monoester. In the process we used Candida antarctica lipase B-displaying Pichia pastoris whole-cells as biocatalyst, glucose as the acyl acceptor and lauric acid as the acyl donor. The product glucose laurate monoester was purified by silica gel column chromatography and preparative liquid chromatography, and identified by liquid chromatography-mass spectrometry. Then we optimized the process from various aspects, such as solvent composition, ratio of dmethyl sulfoxide to 2-Methyl-2-butanol (V/V), catalyst dosage, substrate concentration, water activity and temperature. The optimal reaction conditions were: glucose 0.5 mmol/L, lauric acid 1.0 mmol/L, ratio of 2-Methyl-2-butanol to Dmethyl sulfoxide is 7:3 in 5 mL volume, temperature 60 degrees C, the best initial water activity of whole-cells biocatalyst is 0.11. The maximum glucose conversion could be 48.7% after 72 h.
Assuntos
Biocatálise , Candida , Ésteres , Química , Metabolismo , Proteínas Fúngicas , Engenharia Genética , Glucose , Química , Metabolismo , Lauratos , Química , Metabolismo , Lipase , Genética , Pichia , Genética , Metabolismo , Proteínas Recombinantes , GenéticaRESUMO
Short-chain esters play a significant role in the food industry as flavor and aroma constituents. Candida antarctica lipase B (CALB) is one of the most effective catalysts for organic synthesis. We constructed a CALB-displaying yeast whole-cell biocatalyst and applied it to esterification from caproic acid and ethanol. CALB was fused with the alpha-agglutinin C-terminal and the signal peptide of Glucoamylase in pICAS, a yeast surface display vector, to construct plasmid pICAS-CALB. An extremely Asn-rich linker, named celAL was inserted in the Xho I of pICAS-CALB to construct plasmid pICAS-celAL-CALB. The fused gene was under the control of GAPDH promoter. After incubated at 30 degrees C for 96 h the lipase hydrolytic activity of the yeast whole cells reached a plateau, 26.26 u/(g x dry cell). In nonaqeous media, the yield of 98.0% ethyl hexanoate was obtained after 24 h esterification from caproic acid and ethanol (the molar ratio of caproic acid : ethanol = 1 : 1.25) using lyophilized CALB displaying yeast whole cells.
Assuntos
Biocatálise , Candida , Caproatos , Metabolismo , Clonagem Molecular , Proteínas Fúngicas , Engenharia Genética , Lipase , Genética , Saccharomyces cerevisiae , Genética , MetabolismoRESUMO
HIV-1 gp41 has been successfully anchored on the cell surface of yeast Saccharomyces cerevisiae by yeast cell-surface display systems using His-tag for the detection of protein expression. Gp41 activity has been detected by gp41 monoclonal antibody. The vector for gp41 yeast display has been constructed as follows: the gene-encoding gp41 was amplified by PCR using pMD18T-gp41 as a template, and then inserted into shuttle vector pICAS-His by restriction enzyme digestion. Next, the vectors were introduced into Saccharomyces cerevisiae MT8-1. After cultivation, recombinant cells were immunofluorescence labelled. The bright green cells were observed by the microscopy indicating the proteins have been displayed on the cell surface successfully, flow cytometry convinced that gp41 has been folded correctly on the cell surface. Then different concentrations of initial glucose were used to enhance the expression of protein. gp41 has been expressed by 82.46% yeast cells as the concentration of glucose was 1%. Protein expression was depressed when the concentration was increased.
Assuntos
Humanos , Engenharia Genética , Métodos , Vetores Genéticos , Genética , Metabolismo , Proteína gp41 do Envelope de HIV , Genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão , Genética , Saccharomyces cerevisiae , Biologia Celular , Genética , Metabolismo , Proteínas de Saccharomyces cerevisiae , Genética , Metabolismo , Propriedades de SuperfícieRESUMO
Objective To observe the prophylactic effects of a HSV-2gD2DNA vaccine in guinea pigs challenged with HSV-2strains.Methods Female guinea pigs were divided into3groups with10each,which was immunized intramuscularly with100?g of pc-gD plasmids(recombinant HSV-2DNA vac-cine),or with pcDNA3blank plasmids,with normal saline as control,respectively.Two booster injections were given on day7and day21.Sera were collected for virus neutralization test on day0,day28,and day56.The animals were challenged with HSV-2strain sav intravaginally,and lesions induced on the external genital skin were scored between day1and day21after challenge.Results The titer of neutralizing anti-body to HSV-2was much higher in the sera from animals immunized by pc-gD plasmids than that from ani-mals immunized by pcDNA3blank plasmids or normal saline.Furthermore,the lesion scores on external genital skin were significantly decreased in pc-gD group than those in other two groups with either primary or recurrent infections.Conclusion The constructed gD2vaccine can efficiently protect guinea pigs from genital infection and reduce recurrent infection induced by latent herpes simplex virus.