High-Resolution Mapping of Genome Position Expression Variation in Bacteria & Engineering of Microbial Consortia for Cellulosic Biochemical Production

Consolidated bioprocessing;Bacterial gene expression;Molecular;Cellular and Developmental Biology;Science;Cellular & Molecular Biology

Scholz, ScottSchmidt, Thomas M ;

University of Michigan

关键词: Consolidated bioprocessing;    Bacterial gene expression;    Molecular;    Cellular and Developmental Biology;    Science;    Cellular & Molecular Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/145962/scholz_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

High-resolution mapping of genome position expression variation in bacteria: To elucidate the effect of position in the bacterial genome on gene expression at a high resolution, we have developed a multiplex strategy to construct and analyze a library of genome-integrated reporters in a single mixed population of Escherichia coli. By randomly integrating a standardized barcoded reporter with Tn5 transposase, transcription from over 144,000 reporters across the bacterial genome was tracked simultaneously. High-resolution mapping of reporter transcription revealed large peaks of high transcriptional propensity centered on ribosomal RNA operons that have not been previously detected. Genes for amino acid biosynthesis were specifically enriched in high transcriptional propensity regions, while prophages and mobile genetic elements were enriched in low transcriptional propensity regions, demonstrating that the E. coli chromosome has evolved gene-independent mechanisms for affecting expression from specific regions. The nucleoid associated proteins H-NS and Fis were highly informative of reporter transcription, which shows ~150-fold variation in transcriptional propensity over its entire length.Engineering of microbial consortia for cellulosic biochemical production: Consolidated bioprocessing is a potential breakthrough technology for reducing costs of biochemical production from lignocellulosic biomass. Production of cellulase enzymes, saccharification of lignocellulose and conversion of the resulting sugars into a chemical of interest occur simultaneously within a single bioreactor. In this study, synthetic fungal consortia composed of the cellulolytic fungus Trichoderma reesei and the production specialist Rhizopus delemar demonstrated conversion of microcrystalline cellulose (MCC) and alkaline pre-treated corn stover to fumaric acid in a fully consolidated manner without addition of cellulase enzymes or expensive supplements such as yeast extract. A Titer of 6.87 g/L of fumaric acid, representing 0.17 w/w yield, were produced from 40 g/L MCC with a productivity of 31.8 mg/L/h. In addition, lactic acid was produced from MCC using a fungal consortium with Rhizopus oryzae as the production specialist. These results are proof-of-concept demonstration of engineering synthetic microbial consortia for CBP production of naturally occurring biomolecules. In order to improve the performance of the fumaric acid production CBP system, we developed selective nitrogen delivery as a method to tune the growth of each specialist. By replacing the urease gene of T. reesei with the amdS gene from Aspergillus nidulans, we reassigned the nitrogen utilization capability of T. reeseifrom urea to acetamide to generate the SND strain. Using SND, we were able to tune the production of fumaric acid in the consortia CBP production system by varying Urea:Acetamid ratio. Tuning enable a higher yield of fumaric acid than previously observed.