Development and application of a droplet-based microfluidic high-throughput screening of Pichia pastoris / 生物工程学报

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.

Application of codon optimization strategy in heterologous protein expression / 生物工程学报

Enzymes are widely used in medical and biopharmaceuticals. They can be used not only for various disease treatments, but also clinical diagnosis. The use of microorganisms to express heterologous proteins has become the easiest and fastest way to obtain enzymes. In order to obtain high concentration and high-quality heterologous proteins, a common method is codon optimization of gene sequences. The traditional codon optimization strategy is mainly based on codon bias and GC content, ignoring complex and varied factors such as translational dynamics and metabolic levels. We provide here comprehensive codon optimization strategy based on gene level, transcriptional level, translational level, post-translational level and metabolic level, mainly including codon bias, codon harmonization, codon sensitivity, adjustment of gene sequence structure and some other influencing factors. We also summarize the aspects of strategy content, theoretical support and application. Besides, the advantages and disadvantages of each strategy are also systematically compared, providing an all-round, multi-level and multi-selection optimization strategy for heterogeneous protein expression, and also providing references for the enzyme industry and biopharmaceuticals.

Biologia molecular de leveduras e sua aplicação biotecnológica para estudo de mecanismos celulares do câncer e seu tratamento / Molecular biology of yeast and its biotechnological application to study the cellular mechanisms of cancer and its treatment

A levedura Saccharomyces cerevisiae é o eucarioto mais bem caracterizado quanto às vias metabólicas, celulares e funções de genes. Calcula-se que dos 6.000 genes codificadores de proteínas, pelo menos 3.000 tenham homologia com genes humanos. Além disso, mesmo quando não há homologia direta, os mecanismos moleculares são conservados e em ensaios de complementação de função é possível caracterizar funções análogas em humanos. Leveduras apresentam além de mecanismos conservados, diversas semelhanças metabólicas com células tumorais; talvez a mais marcante delas seja a repressão catabólica por glicose. Esse fenômeno causa uma superativação da via glicolítica e inibição da cadeia respiratória, o que lembra muito o efeito Warburg apresentado pelas células tumorais. Esse comportamento metabólico tem relação estreita com a homeostase redox celular. Estes fatores mostram o quanto à caracterização dessas células fúngicas pode ser aplicada ao entendimento e tratamento do câncer. Mesmo assim, 20 anos após o término do sequenciamento do genoma de S. cerevisiae, mais de 1.000 genes continuam anotados como não caracterizados. Além disso, as leveduras têm papel fundamental na produção biotecnológica de insumos farmacêuticos. Nesse texto sistematizado relato as contribuições dos meus estudos, juntamente com os resultados do meu grupo de pesquisa, usando a biologia molecular das leveduras, para responder questões aplicadas à compreensão de funções celulares (com destaque à resposta antioxidante), mecanismos moleculares de resposta aos antitumorais e produção de biofármacos. Além de modelo celular, as leveduras representam excelentes plataformas para expressão heteróloga de proteínas de interesse Biotecnológico-Farmacêutico

The yeast Saccharomyces cerevisiae is the most well characterized eukaryote for metabolic, cellular and gene functions. It is estimated that of the 6,000 protein-encoding genes, at least 3,000 have homology to human genes. Moreover, even when there is no direct homology, the molecular mechanisms are conserved and through function complementation assays it is possible to characterize analogous functions in humans. Yeasts present, in addition to conserved mechanisms, diverse metabolic similarities with tumor cells; perhaps the most remarkable of them is the catabolic repression by glucose. This phenomenon causes an over activation of the glycolytic pathway and inhibition of the respiratory chain, which strongly resembles the Warburg effect presented by tumor cells. This metabolic behavior is closely related to cellular redox homeostasis. These factors show how much the characterization of these fungal cells can be applied to the understanding and treatment of cancer. Even so, 20 years after the end of the sequencing of the S. cerevisiae genome, more than 1,000 genes remain annotated as uncharacterized. In addition, yeasts play a key role in the biotechnological production of pharmaceutical inputs. In this systematized text I show the contributions of my studies, together with the results of my research group, using molecular biology of yeast to answer questions applied to understanding cellular functions (with emphasis on the antioxidant response), molecular response mechanisms to antitumor drugs and biopharmaceutical production. In addition to cellular model, yeasts represent excellent platforms for the heterologous expression of proteins of Biotechnological-Pharmaceutical interes

Probiotic characterization of lactic acid bacteria isolated from infants feces and its application for the expression of green fluorescent protein

Aims: In this study, lactic acid bacteria (LAB) were isolated from 42 healthy infants and determined for probiotic properties. Twelve LAB isolates with potential probiotic properties were selected and screened for their feasibility of heterologous protein expression by selection of erythromycin sensitive isolates. Methodology and results: One of eleven erythromycin-sensitive LAB isolates identified and designated as Lactobacillus fermentum 47-7 was able to acquire and stable maintain the Escherichia coli-Lactobacillus shuttle vector, pRCEID-LC13.9. Further electrotransformation of L. fermentum 47-7 with the recombinant pLC13.9:LDH-PRO1:GFPuv containing green fluorescent protein (GFP) gene found that recombinant L. fermentum can express GFP. Conclusion, significance and impact of study: The probiotic L. fermentum isolate can be used as host for expression of heterologous proteins and could possibly be further developed as the alternate oral delivery system for various biomolecules for biotechnological application.