Transcriptomic analysis of interaction dynamics between meju Zygomycetes and Aspergillus = 메주에서 분리한 Zygomycetes와 Aspergillus 상호작용 역학에 대한 전사체 분석

다국어 초록 (Multilingual Abstract)

The spontaneous fermentation of meju, a traditional Korean soybean brick, involves complex microbial successions dominated first by Zygomycetes (e.g., Mucor) and later by Aspergillus species. However, the molecular basis of these ecological transitions remains poorly understood. This study employed a non-contact co- culture system using Aspergillus oryzae MJS14 and Mucor circinelloides KACC40262—both isolated from meju—to elucidate interspecies interaction dynamics through time-resolved transcriptomic profiling at 18, 36, and 54 h. RNA-seq analysis revealed a temporally phased interaction. In early stages (18 h), A. oryzae exhibited strong transcriptional suppression of ABC transporters and redox enzymes, suggesting early inhibition by Mucor-derived metabolites. By 36 h, A. oryzae activated chitosanase (GH75, GH18) genes targeting Mucor’s chitosan- rich cell wall, along with secondary metabolite clusters for heptelidic acid and 8- methyldiaporthin, indicating enzymatic and chemical antagonism. At 54 h, kojic acid biosynthetic genes (kojA, kojR, kojT) were highly upregulated, implying a late- phase strategy of metal ion competition via iron and zinc chelation. Conversely, M. circinelloides exhibited adaptive metabolic responses characterized by increased carbohydrate utilization, acid metabolism, and siderophore biosynthesis. At 36 h, genes for glycolysis, amino/organic-acid metabolism, and the NRPS- independent siderophore (rhizoferrin) pathway were strongly induced. Concurrently, carotenoid (C40 tetraterpene) and glycan remodeling clusters were activated, reflecting oxidative-stress mitigation and surface defense remodeling. Together, these findings reveal a sequential antagonistic interaction: Mucor dominates early through metabolic suppression, Aspergillus counterattacks mid- phase via CWDEs / antifungal metabolites, and the interaction culminates in nutrient and metal competition mediated by kojic acid and Rhizoferrin. This transcriptomic insight provides a mechanistic framework for understanding fungal succession and ecological balance during meju fermentation. Keywords: meju, Aspergillus oryzae, Mucor circinelloides, RNA-seq, co-culture, secondary metabolites, chitosanase, kojic acid, rhizoferrin, fungal interaction

국문 초록 (Abstract)

메주의 자연발효는 초기에는 Zygomycetes (예: Mucor)가 우점하고 이후에는 Aspergillus 종이 우점하는 복잡한 미생물 천이를 동반한다. 그러나 이러한 생태학적 전환을 유도하는 분자적 기전은 아직 충분히 규명되지 않았다. 본 연구에서는 메주에서 분리한 Aspergillus oryzae MJS14와 Mucor circinelloides KACC40262를 비접촉 공배양(non-contact co-culture) 시스템에서 배양하고, 18, 36, 54 h에서 시간별 전사체(RNA-seq)를 분석하여 종간 상호작용의 동역학을 규명하였다.
RNA-seq 결과, 상호작용은 시간에 따라 단계적으로 진행되었다. 초기(18 h)에는 A. oryzae에서 ABC 수송체 및 산화환원 효소 관련 유전자의 전사가 강하게 억제되어, Mucor 유래 대사산물에 의한 초기 저해 가능성을 시사하였다. 중기(36 h)에는 A. oryzae가 Mucor의 키토산 기반 세포벽을 표적으로 하는 키토사나아제(GH75, GH18) 유전자를 활성화하고, heptelidic acid 및 8-methyldiaporthin 관련 이차대사 유전자 클러스터 발현을 증가시켜 효소적·화학적 길항 작용이 강화됨을 확인하였다. 후기(54 h)에는 kojic acid 생합성 유전자(kojA, kojR, kojT)가 크게 상향 조절되어, 철 및 아연 킬레이션을 통한 금속 이온 경쟁 전략으로 전환됨을 시사하였다.
반대로 M. circinelloides는 탄수화물 이용 증가, 산 대사, 시데로포어 생합성 등 적응적 대사 반응을 보였다. 특히 36 h에서 해당 균주의 해당과정(glycolysis), 아미노산/유기산 대사 관련 유전자와 NRPS-비의존성 시데로포어(rhizoferrin) 경로 유전자 발현이 강하게 유도되었다. 동시에 카로티노이드(C40 tetraterpene) 및 당질(glycan) 리모델링 관련 클러스터가 활성화되어, 산화스트레스 완화 및 표면 방어 구조 재편이 진행됨을 나타냈다.
종합하면, 본 연구는 “초기 Mucor의 대사적 억제 → 중기 Aspergillus의 세포벽 분해효소 및 항진균 이차대사산물에 의한 반격 → 후기 kojic acid와 rhizoferrin에 기반한 영양·금속 경쟁”으로 이어지는 순차적 길항 상호작용 모델을 제시한다. 이러한 전사체 기반 기계적 통찰은 메주 발효에서 관찰되는 곰팡이 천이와 생태적 균형 형성의 분자적 기반을 이해하기 위한 틀을 제공한다.

목차 (Table of Contents)

• Ⅰ INTRODUCTION 1
• Ⅱ MATERIALS AND METHODS 5
• 1 Fungal strains 5
• 2 Culture conditions 5
• 2.1 Fungal activation 5
• 2.2 Non-physical contact co-culture of A. oryzae and M. circinelloides 5
• 3 RNA extraction and cDNA synthesis for transcriptomic analysis 6
• 4 Bioinformatics pipeline 8
• 4.1 Whole Genome Sequencing(WGS) and annotation of M. circinelloides KACC40262 8
• 4.2 Sequence trimming 8
• 4.3. Read mapping and quantitative counting 8
• 4.4. Differentially Expressed Genes(DEGs) filtering and annotation mapping 9
• 4.5. Gene ontology(GO) and GSEA(Gene set enrichment analysis) visualization 10
• 4.6. Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis 10
• 4.7. Carbohydrate enzymes(CAZyme) analysis and normalization 11
• 4.8. Transcripts expression analysis of secondary metabolites and Biosynthetic Gene Clusters(BGCs) 12
• 4.9. γ-glutamyl transpeptidases(GGT) and leucine aminopeptidases(LAP) genes expression during co-culture 12
• Ⅲ RESULTS 13
• 1 Summary of sequencing data 13
• 2 Temporal gene expression and overall results in A. oryzae 15
• 2.1. Principal Component Analysis(PCA) of A. oryzae during co-culture 15
• 2.2. DEG analysis in A. oryzae under interactions between A. oryzae and M. circinelloides 17
• 2.3.GO enrichment reveals temporal transitions in defense and metabolic strategy 19
• 2.4. Carbohydrate enzyme (CAZyme) profiling reveals polysaccharide-specific remodeling and antagonistic responses 21
• 2.5.KEGG analysis reveals dynamic metabolic and detoxification shifts in response to interactions 23
• 2.6. Analysis of secondary metabolite Biosynthesis Gene Clusters (BGCs) of A. oryzae based on the MIBiG database 25
• 3 M. circinelloides whole genome sequencing data comprehensive analysis 27
• 4 Temporal gene expression and overall results in M. circinelloides KACC40262 29
• 4.1. PCA of M. circinelloides during co-culture 29
• 4.2. DEG analysis in M. circinelloides under between A. oryzae and M. circinelloides 31
• 4.3. Top10 most significant up and down regulated genes of M. circinelloides 33
• 4.4. GO enrichment analysis and Acid term expression analysis of DEGs in M. circinelloides under interaction with A. oryzae 35
• 4.5. Carbohydrate enzyme(CAZyme) profiling reveals polysaccharide-specific remodeling and antagonistic responses 37
• 4.6. KEGG analysis reveals dynamic metabolic and detoxification shifts in response to interactions. 39
• 4.7. anti-SMASH predicted cluster expression analysis of M. circinelloides co-culture with A. oryzae. 41
• 5 Expression changes of GGT and LAP genes during co-culture 43
• Ⅳ DISCUSSION 45
• Ⅴ REFERENCES 52