Dataset Information

Comparative RNAseq analyses between P. acidipropionici WGS7 and P. acidipropionici ATCC 55737

ABSTRACT: Propionic acid (PA) is a three-carbon molecule commonly used as a food preservative and increasingly, as a precursor for the synthesis of monomers. Propionibacterium spp. are the best biological producers of PA. In fact, a recent report showed that if a yield of 0.6 g/g is achieved, biological production of PA would be economically competitive with petrochemical production. To achieve that yield, a library of Propionibacterium strains was used to generate a new strain that can achieve the commercially desirable yield from sucrose. The genome of the new strain was sequenced and a series of SNPs were found to be responsible for the improved phenotype. Using a combination of transcriptomics the relevant mutations were expounded. Differential RNA-sequencing between the wild-type and the mutant strain identified the relevant genomic changes responsible for the PA yield improvement. Notably, an increase in the specific consumption rate of sucrose was attributed to a SNP in the promoter region of a sugar transporter. Similarly, a mutation in a polar amino acid transporter improved acid tolerance, and an improvement in the electron transport system rewired the metabolism to yield an improved PA phenotype. These changes resulted in a lower acetic acid by-product generation and a higher PA yield. The productivity of the new strain was augmented with the design of a fed-batch process to achieve titres of 70 g/L through the use of a mathematically design fed-strategy.

ORGANISM(S): Acidipropionibacterium acidipropionici

PROVIDER: GSE86950 | GEO | 2018/12/14

SECONDARY ACCESSION(S): PRJNA343046

REPOSITORIES: GEO

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Project description:Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-H+ symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9 % higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5’ coding sequences of SUC2, resulting in relocation of invertase to the cytosol. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 95 generations of anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11 % higher ethanol yield on sucrose in chemostat and batch cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. Deletion of AGT1, which had been duplicated during laboratory evolution, restored the growth characteristics of the unevolved iSUC2 strain. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes. The goal of the present study was to investigate whether a relocation of sucrose hydrolysis to the cytosol can be used to improve ethanol yields on sucrose and which additional steps may be required to improve sucrose utilization by strains that only express intracellular invertase. To this end, the SUC2 gene was modified to cause an exclusive intracellular localization. Growth and product formation by the engineered strain were compared with that of the parental strain in anaerobic sucrose-limited chemostat cultures. Subsequently, evolutionary engineering was used to improve sucrose uptake kinetics and an evolved strain was characterized for growth and product formation in chemostat cultures. Transcriptome analysis and gene deletion studies were used to identify genetic changes in the evolved strain that contribute to its improved sucrose-uptake kinetics.

Dataset Information

Comparative RNAseq analyses between P. acidipropionici WGS7 and P. acidipropionici ATCC 55737

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