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Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion.

ABSTRACT: With emerging interests in heterologous production of proteins such as antibodies, growth factors, nanobodies, high-quality protein food ingredients, etc. the demand for efficient production hosts increases. Corynebacterium glutamicum is an attractive industrial host with great secretion capacity to produce therapeutics. It lacks extracellular protease and endotoxin activities and easily achieves high cell density. Therefore, this study focuses on improving protein production and secretion in C. glutamicum with the use of droplet-based microfluidic (DBM) high throughput screening. A library of C. glutamicum secreting β-glucosidase was generated using chemical mutagenesis coupled with DBM screening of 200,000 mutants in just 20 min. Among 100 recovered mutants, 16 mutants exhibited enhanced enzyme secretion capacity, 13 of which had unique mutation profiles. Whole-genome analysis showed that approximately 50-150 SNVs had occurred on the chromosome per mutant. Functional enrichment analysis of genes with non-synonymous mutations showed overrepresentation of genes involved in protein synthesis and secretion relevant biological processes, such as DNA and ribosome RNA synthesis, protein secretion and energy turnover. Two mutants JCMT1 and JCMT8 exhibited the highest secretion with a six and a fivefold increase in the β-glucosidase activity in the supernatant, respectively, relative to the reference strain JC0190. After plasmid curing, a new plasmid with the gene encoding α-amylase was cloned into these two mutants. The new strains SB024 and SB025 also exhibited a five and a sixfold increase in α-amylase activity in the supernatant, respectively, relative to the reference strain SB023. The results demonstrate how DBM screening can serve as a powerful development tool to improve cell factories for the production and secretion of heterologous proteins.

SUBMITTER: Balasubramanian S

PROVIDER: S-EPMC8137953 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Succinate has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. However, efficient methods for the production of succinate from lignocellulosic feedstock were rarely reported. Nevertheless, Corynebacterium glutamicum was engineered to efficiently produce succinate from glucose in our previous study. Results:In this work, C. glutamicum was engineered for efficient succinate production from lignocellulosic hydrolysate. First, xylose utilization of C. glutamicum was optimized by heterologous expression of xylA and xylB genes from different sources. Next, xylA and xylB from Xanthomonas campestris were selected among four candidates to accelerate xylose consumption and cell growth. Subsequently, the optimal xylA and xylB were co-expressed in C. glutamicum strain SAZ3 (?ldhA?pta?pqo?catPsod-ppcPsod-pyc) along with genes encoding pyruvate carboxylase, citrate synthase, and a succinate exporter to achieve succinate production from xylose in a two-stage fermentation process. Xylose utilization and succinate production were further improved by overexpressing the endogenous tkt and tal genes and introducing araE from Bacillus subtilis. The final strain C. glutamicum CGS5 showed an excellent ability to produce succinate in two-stage fermentations by co-utilizing a glucose-xylose mixture under anaerobic conditions. A succinate titer of 98.6 g L-1 was produced from corn stalk hydrolysate with a yield of 0.87 g/g total substrates and a productivity of 4.29 g L-1 h-1 during the anaerobic stage. Conclusion:This work introduces an efficient process for the bioconversion of biomass into succinate using a thoroughly engineered strain of C. glutamicum. To the best of our knowledge, this is the highest titer of succinate produced from non-food lignocellulosic feedstock, which highlights that the biosafety level 1 microorganism C. glutamicum is a promising platform for the envisioned lignocellulosic biorefinery.

Project description:Due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. This strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. Of these platforms, metabolic profiling has the strongest correlation with the phenotype. We previously published a high-throughput metabolic profiling method for C. glutamicum as well as the automatic GC/MS processing software MetaboliteDetector. Here, we added a high-throughput transposon insertion determination for our C. glutamicum mutant library. The combination of these methods allows the parallel analysis of genotype/phenotype correlations for a large number of mutants. In a pilot project we analyzed the insertion points of 722 transposon mutants and found that 36% of the affected genes have unknown functions. This underlines the need for further information gathered by high-throughput techniques. We therefore measured the metabolic profiles of 258 randomly chosen mutants. The MetaboliteDetector software processed this large amount of GC/MS data within a few hours with a low relative error of 11.5% for technical replicates. Pairwise correlation analysis of metabolites over all genotypes showed dependencies of known and unknown metabolites. For a first insight into this large data set, a screening for interesting mutants was done by a pattern search, focusing on mutants with changes in specific pathways. We show that our transposon mutant library is not biased with respect to insertion points. A comparison of the results for specific mutants with previously published metabolic results on a deletion mutant of the same gene confirmed the concept of high-throughput metabolic profiling. Altogether the described method could be applied to whole mutant libraries and thereby help to gain comprehensive information about genes with unknown, hypothetical and known functions.

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Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion.

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