Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions inPichia pastorisin methanol-free media

Demir I., ÇALIK P.

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, cilt.104, sa.19, ss.8381-8397, 2020 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 104 Sayı: 19
  • Basım Tarihi: 2020
  • Doi Numarası: 10.1007/s00253-020-10796-5
  • Dergi Adı: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, ABI/INFORM, Agricultural & Environmental Science Database, Applied Science & Technology Source, Aqualine, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Chemical Abstracts Core, Compendex, Computer & Applied Sciences, EMBASE, Environment Index, Food Science & Technology Abstracts, Geobase, MEDLINE, Pollution Abstracts, Veterinary Science Database
  • Sayfa Sayıları: ss.8381-8397
  • Anahtar Kelimeler: Double-promoter expression system (DPES), Deregulated gene expression, Engineered promoter, Alcohol oxidase 1(AOX1) promoter, Methanol-free media, Pichia pastoris(Komagataella phaffii), RECOMBINANT PROTEIN-PRODUCTION, SYNTHETIC CORE PROMOTERS, PICHIA-PASTORIS, BETA-MANNANASE, GAP, FERMENTATION
  • Orta Doğu Teknik Üniversitesi Adresli: Evet

Özet

Double-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants P(ADH2-Cat8-L2)and P(mAOX1)and with the naturally occurring promoter P(GAP)to enhance and upregulate-deregulated gene expressions inPichia pastorisin methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were expressed under P(ADH2-Cat8-L2)and P(mAOX1)or P-GAP, respectively, enabling the determination of the transcription period and strength of each constituent in the DPESs. We determined fluorescent protein expressions in batch cultivations on 2% (v/v) ethanol, excess glucose, and excess glycerol, and compared them with single-promoter expression systems constructed with PADH2-Cat8-L2, P-mAOX1, and P-GAP. The transcription- and expression-upregulation power of bifunctional DPESs was higher than that of twin DPESs (two-copy expression systems). Our findings answer long-standing questions regarding the high- (or multi-) copy clone results in the literature. Our first conclusion is that increasing identical components in the DPES architectures linearly increases the concentrations ofcis-acting DNA sites and increases the demand for key transcription factors (TFs) that perturb their good coupling of supply and demand. The next is that the synthesis of some amino acids may create bottleneck(s) as rate-limiting amino acid(s) in recombinant protein synthesis. With bifunctional DPESs, each constituent upregulated the transcription and increased the expression and reduced the demand for the same TF(s) in the generation of novel regulatory circuits, due to the increased number of nonidenticalcis-acting DNA sites. We tested superior DPES performances in extracellular human growth hormone (rhGH) production. Thereby, the indications related to the rate-limiting amino acids were verified. Compared with its constituents P(ADH2-Cat8-L2)and P-mAOX1, the bifunctional DPES(4)enhanced rhGH production by 1.44- and 2.02-fold, respectively. The DPES design method, with its constraint and parameters, enables the generation of promising r-protein production platforms with high impact on industrial-scale production processes and opens up new avenues for research in yeasts.