Engineering of alcohol dehydrogenase 2 hybrid‐promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol

Abstract

The aim of this work is to increase recombinant protein expression in Pichia pastoris over the ethanol utilization pathway under novel-engineered promoter variants (NEPVs) of alcohol dehydrogenase 2 promoter (P ADH2 ) through the generation of novel regulatory circuits. The NEPVs were designed by engineering of transcription factor binding sites (TFBSs) determined by in silico analyses and manual curation systematically, by (a) single-handedly replacement of specified TFBSs with synthetic motifs for Mxr1, Cat8, and Aca1 binding, and synthetic TATA-box integration; and, (b) nucleosome optimization. P ADH2-Cat8-L2 and P ADH2-Cat8-L1 designed by the integration of synthetic Cat8 binding sites were superior, and then P ADH2-NucOpt .Compared to that with P ADH2 at t = 20 hr of the fermentations, P ADH2-Cat8-L2 allowed the highest increase in enhanced green fluorescent protein expression as 4.8-fold on ethanol and 3.8-fold on methanol; and, P ADH2-NucOpt upregulated the expression 1.5-fold on ethanol and enhanced 3.2-fold on methanol. Using the superior two tools, Cat8-L2 and NucOpt, we designed P ADH2-NucOpt-Cat8-L2 . With P ADH2-NucOpt-Cat8-L2 , the expression in the fermentation of ethanol was upregulated 3.7-fold that is distinctly higher than that with P ADH2-NucOpt but lower than that with P ADH2-Cat8-L2 ; while on methanol compared to that with P ADH2 , the expression was enhanced 8.8-fold. Extracellular recombinant human serum albumin production was also studied with P ADH2-Cat8-L2 and P ADH2-NucOpt , and average recombinant human serum albumin yield (Y P/X ) on ethanol was 1.13 and 0.38 mg/g WCW, respectively; whereas with P ADH2 , Y P/X was 0.26 mg/g WCW . We conclude that as upregulation of transcription and enhanced expression correlate with the sequence of synthetic motifs and their location in the hybrid-promoter architectures of NEPVs in coordination with trans-acting factors, which are the design parameters in the engineering of binding sites; the NEPVs generated promising recombinant protein production platforms with a high impact on industrial scale production processes, as well as would open up new avenues for research in P. pastoris. K E Y W O R D S alcohol dehydrogenase 2 promoter, Cat8, hybrid-promoter architecture, novel-engineered promoter variant, nucleosome optimization, Pichia pastoris (Komagataella phaffii), synthetic binding site

Identification of major ADH genes in ethanol metabolism of Pichia pastoris

“…The ADH2 gene displayed a similar expression profile with ADH900 on glucose, glycerol, and methanol, but exhibited a significant increase in response to ethanol (Figure ). This result points to ethanol induction of the ADH2 gene, which confirms previous findings (Ergün et al, ; Karaoglan et al, , ). On the other hand, it is also worth mentioning that when the cells were grown on glucose, Adh2 activity is detected as soon as ethanol begins to be produced even in the presence of glucose (Figure ).…”

“…On the other hand, a sharp increase of the ADH2 expression level was observed in ethanol‐grown cells (Figure ). Distinct ethanol induction of the ADH2 gene was previously confirmed with recombinant protein production under the ADH2 promoter (Ergün et al, ; Karaoglan et al, ). Because the ADH2 encodes the enzyme which catalyzes the first reaction of ethanol utilization pathway, such a strong activation of the ADH2 expression is fairly expected in cells grown in ethanol.…”

“…The highest yield was determined in ADH2 promoter‐controlled productions suggesting it a promising alternative for recombinant protein production. The regulation mechanism of the promoter region has then been investigated by generating new promoter variants in terms of transcription factor binding sites with the purpose of increasing the recombinant protein expression (Ergün, Gasser, Mattanovich, & Çalık, ).…”

Function and regulation of an aldehyde dehydrogenase essential for ethanol and methanol metabolism of the yeast,Komagataella phaffii

“…GenBank accession numbers are shown in parentheses. * denoted as ADH2 (27,46,45) or ADH3 (26). @ denoted as ADH6 (45) # denoted as ALD5 (45) + denoted as ALD4 (27 and 45)…”

Transcriptomic Analysis of Pichia pastoris (Komagataella phaffii) GS115 During Heterologous Protein Production Using a High-Cell-Density Fed-Batch Cultivation Strategy

“…Thus, it is imperative that further efforts are made to improve levels of expression exhibited by the analyzed strain. With this in mind, many researchers have conducted experiments that have resulted in the improvement of protein stability through rational design (Han et al, 2017(Han et al, , 2018, the development of strong promoters (Portela et al, 2018;Prielhofer et al, 2018;Ergun et al, 2019;, the optimization of protein secretion (Barrero et al, 2018;Nguyen et al, 2018), the alteration of protein glycation in P. pastoris (Pekarsky et al, 2018), and co-expression of transcription factors (Chang et al, 2018;Dey et al, 2018). Furthermore, increasing productivity during scale-up of processes largely depends on trial and Abbreviations: 6PG, gluconate-6P; CTS, citrate synthase; DAK, triose/dihydroxyacetone kinase; DAS, dihydroxyacetone synthase; DHA, dihydroxyacetone; DHAP, dihydroxyacetone-P; DLD, dihydrolipoamide dehydrogenase; E4P, erytgrose-4P; ENOI, enolase I; F1,6BP, fructose-1, 6-P2; F6P, fructose-6P; FBAII, fructose-bisphosphate aldolase, class II; FBPI, fructose-1,6-bisphosphatase I; FCH, S-formylglutathione hydrolase; FDH, formate dehydrogenase; FLD1, S-(hydroxymethyl)glutathione dehydrogenase; Form, formaldehyde; FUM, fumarate; G1,3P, glycerate-1,3P2; G2P, glycerate-2P; G3P, glycerate-3P; G6P, glucose-6P; GAP, glyceraldehyde-3P; GAPD, glyceraldehyde 3-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; GS-CH 2 OH, S-hydroxymethylglutathione; GS-CHO, S-formylglutathione; ISO, isocitrate; MAL, malate; MDH, malate dehydrogenase; ODE, 2-oxoglutarate dehydrogenase E2; OXA, oxaloacetate; OXAL, oxalosuccinate; PDE, pyruvate dehydrogenase E; PEP, phosphoenolpyrvate; PEX, peroxisomal; PGK, phosphoglycerate kinase; R5P, ribose-5P; RKI, ribose-5-phosphate ketol-isomerase; Rul5P, ribulose-5P; S7P, sedoheptulose-7p; SCS1, succinyl-CoA synthetase 1; SCS2, succinyl-CoA synthetase 2; SDH, succinate dehydrogenase; SUC, succinate; TAL, transaldolase; TK, transketolase; X5P, xylulose-5P.…”