Transcriptome analysis of Rhizopus oryzae seed pellet formation using triethanolamine

Abstract

Rhizopus oryzae (R. oryzae) can effectively produce organic acids, and its pellet formation in seed cultures has been shown to significantly enhance subsequent fermentation processes. Despite advances in strain development, simple and effective methods for inducing pellet morphology and a basic understanding of the mechanisms controlling this process could facilitate substantial increases in efficiency and product output. Here, we report that 1.5% triethanolamine (TEOA) in seed culture medium can activate the growth of R. oryzae spores in compact and uniform pellets which is optimal for fermentation conditions. Analysis of fermentation kinetics showed that the production of fumaric and L-malic acid increases 293% and 177%, respectively. Transcriptomic analysis revealed that exposure of R. oryzae to 1.5% TEOA during the seed culture activated the phosphatidylinositol and mitogen-activated protein kinase signaling pathways. Theses pathways subsequently stimulated the downstream carbohydrate-active synthases and hydrolases that required for cell wall component synthesis and reconstruction. Our results thus provide insight into the regulatory pathways controlling pellet morphology germane to the viability of seed cultures, and provide valuable reference data for subsequent optimization of organic acid fermentation by R. oryzae.

Keywords: Organic acid; R. oryzae; Seed pellet formation; Transcriptomic analysis; Triethanolamine.

Fig. 1

Scheme of the experimental strategy and analysis of R. oryzae pellet formation induced by surfactants. A Illustration of R. oryzae seed pellet formation induced by triethanolamine (TEOA). B Fermentation assay of R. oryzae with different seed morphologies. C Transcriptomic analysis revealing the probable mechanism of pellet formation

Fig. 2

Optimization of TEOA addition in seed culture medium. A Different R. oryzae seed morphologies induced by different concentration of TEOA. B Effects of the triethanolamine concentration on R. oryzae seed pellet diameter. C SEM images of R. oryzae after seed culture with or without 1.5% TEOA. D Effects of the triethanolamine concentration on the CDW of R. oryzae seed cultures. Data represent means ± SD of three independent replicates. Statistical significance was determined by Student’s t test (n = 3). *p < 0.05, **p < 0.01

Fig. 3

Fermentation kinetics of R. oryzae with different morphologies formed by adding different dosages of TEOA in seed culture medium. A Glucose concentration in fermentation medium. B CDW. C Fumaric acid production. D Malic acid production. Data represent means ± SD of three independent replicates. Statistical significance was determined by Student’s t test (n = 3). * p < 0.05, ** p < 0.01

Fig. 4

Transcriptomic profiling and annotation of DEGs between the seed culture medium with triethanolamine (SCM_TEOA)-treated group and the seed culture medium with surfactant-free (SCM_con) group. A score plots of the transcriptome of the SCM_TEOA group compared to the SCM_con group. B Volcano plots of the transcriptome of the SCM_TEOA group compared to the SCM_con group. C Up- and down-regulated DEGs in the SCM_TEOA compared to the SCM_con group. D GO annotation of DEGs in the SCM_TEOA group compared to the SCM_con group. E KEGG annotation of the DEGs in the SCM_TEOA group compared to the SCM_con group

Fig. 5

Proposed cell signal transduction pathway regulating cell wall reconstruction during TOEA-induced pellet formation