Discovery of a rhamnose utilization pathway and rhamnose-inducible promoters in Pichia pastoris

Liu, Bo; Zhang, Yuwei; Zhang, Xue; Yan, Chengliang; Zhang, Yuhong; Xu, Xinxin; Zhang, Wei

doi:10.1038/srep27352

Cited by 27 publications

(25 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, to achieve specificity, we considered it unlikely that the originally described CGG-X 11 -CCG site [43], which is an exact replicate of the Gal4p responsive element, is the full recognition element. The extra T nucleotide we found was also present in the EMSA constructs used by Pardo and Orejas [43], in at least one half-site of the TCGG-X 11 -CGC motif found to be important for strong l -Rha-dependent induction of lra3 in Pichia pastoris [56], and in the shorter binding motif identified by Weirauch et al [46] (http://cisbp.ccbr.utoronto.ca/index.php). Therefore, we think it likely that the PDR-1/RhaR responsive element is represented by the TCGG-X 11 -CCGA motif.…”

Section: Discussionmentioning

confidence: 97%

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa

Thieme

Dietschmann

et al. 2017

Biotechnol Biofuels

View full text Add to dashboard Cite

BackgroundPectin is an abundant component in many fruit and vegetable wastes and could therefore be an excellent resource for biorefinery, but is currently underutilized. Fungal pectinases already play a crucial role for industrial purposes, such as for foodstuff processing. However, the regulation of pectinase gene expression is still poorly understood. For an optimal utilization of plant biomass for biorefinery and biofuel production, a detailed analysis of the underlying regulatory mechanisms is warranted. In this study, we applied the genetic resources of the filamentous ascomycete species Neurospora crassa to screen for transcription factors that play a major role in pectinase induction.ResultsThe pectin degradation regulator-1 (PDR-1) was identified through a transcription factor mutant screen in N. crassa. The Δpdr-1 mutant exhibited a severe growth defect on pectin and all tested pectin-related poly- and monosaccharides. Biochemical as well as transcriptional analyses of WT and the Δpdr-1 mutant revealed that while PDR-1-mediated gene induction was dependent on the presence of l-rhamnose, it also strongly affected the degradation of the homogalacturonan backbone. The expression of the endo-polygalacturonase gh28-1 was greatly reduced in the Δpdr-1 mutant, while the expression levels of all pectate lyase genes increased. Moreover, a pdr-1 overexpression strain displayed substantially increased pectinase production. Promoter analysis of the PDR-1 regulon allowed refinement of the putative PDR-1 DNA-binding motif.ConclusionsPDR-1 is highly conserved in filamentous ascomycete fungi and is present in many pathogenic and industrially important fungi. Our data demonstrate that the function of PDR-1 in N. crassa combines features of two recently described transcription factors in Aspergillus niger (RhaR) and Botrytis cinerea (GaaR). The results presented in this study contribute to a broader understanding of how pectin degradation is orchestrated in filamentous fungi and how it could be manipulated for optimized pectinase production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0807-z) contains supplementary material, which is available to authorized users.

show abstract

Section: Discussionmentioning

confidence: 97%

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa

Thieme

Dietschmann

et al. 2017

Biotechnol Biofuels

View full text Add to dashboard Cite

show abstract

“…A previous study preliminarily investigated several rhamnose-inducible promoters in Pichia pastoris . Among them, the transcription activities of two promoters, P LRA3 and P LRA4 , were higher than those of other promoters, including PAS_chr4_0338 promoter (P LRA1 ), PAS_chr4_0339 promoter (P LRA2 ), PAS_chr4_0340 promoter (P LRAR ) ( Liu et al, 2016 ), which indicated that LRA3 and LRA4 were rate-determining step enzymes related to rhamnose metabolism. Recombinant proteins under the control of the two promoters, especially P LRA3 , could be efficiently produced using rhamnose as the inducer ( Liu et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Decreased Rhamnose Metabolic Flux Improved Production of Target Proteins and Cell Flocculation in Pichia pastoris

Yan

Zhao

et al. 2018

Front. Microbiol.

Self Cite

View full text Add to dashboard Cite

Previously, several genes, including LRA1–LRA4 and LRAR, involved in rhamnose utilization pathway, were discovered in Pichia pastoris GS115; among them, LRA3 and LRA4 were considered as key rate-determining step enzymes. A P. pastoris expression platform based on the strong rhamnose-inducible promoter PLRA3 did not meet the demands of industrial application due to poor production of recombinant proteins. To enhance recombinant protein production of this expression platform, a genetically engineered strain, P. pastoris GS115m, with decreased rhamnose metabolic flux was developed from P. pastoris GS115 by replacement of the rhamnose-inducible promoter PLRA4 with another much weaker rhamnose-inducible promoter, PLRA2. Grown in MRH and YPR media using rhamnose as the main carbon source, the engineered strain showed decreased growth rate and maximal biomass compared with the parental strain. More importantly, grown in rhamnose-containing MRH and YPR media, the recombinant engineered strain harboring a β-galactosidase gene lacB, whose expression was regulated by rhamnose-inducible PLRA3, yielded substantial increases, of 2.5- and 1.5-fold, respectively, in target protein production over the parental strain. Additionally, grown in MRH and YPR media, the engineered strain had remarkable cell flocculation and rapid sedimentation with the increasing of cell density, providing an effective and convenient separation of the fermentation supernatant from strain cells. The engineered strain is a promising expression host for industrial production of target proteins due to its advantages over the parental strain as follows: (i) improved production of recombinant proteins, and (ii) remarkable cell flocculation and rapid sedimentation.

show abstract

“…The cells were collected using centrifugation (12,000g) at 4 °C for 4 min and stored at − 80 °C before total RNA extraction. Total RNA extraction and trace DNA removal were performed according to previously described methods (Liu et al 2016). RNA concentration was determined with a Qubit ® 2.0 fluorometer (Life Technologies, CA, USA), and the quality was checked using an Agilent RNA 6000 Nano kit combined with an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA).…”

Section: Production Determination Of Recombinant Proteinmentioning

confidence: 99%

“…Simultaneously, the promoters of LRA3 and LRA4, P LRA3 and P LRA4 , were identified as two strong rhamnoseinducible promoters (Jiao et al 2019;Liu et al 2016). Subsequently, a Pichia expression system based on P LRA3 , designated as the P LRA3 system, was developed using rhamnose as the inducer.…”

Section: Introductionmentioning

confidence: 99%

Decreased expression of LRA4, a key gene involved in rhamnose metabolism, caused up-regulated expression of the genes in this pathway and autophagy in Pichia pastoris

et al. 2020

Self Cite

View full text Add to dashboard Cite

In a previous study, we developed Pichia pastoris GS115m, an engineered strain with decreased expression of one key gene, LRA4, in rhamnose metabolism. P. pastoris GS115m/LacB was subsequently constructed via introducing a β-galactosidase gene, LacB, under the control of rhamnose-inducible P LRA3 into P. pastoris GS115m. P. pastoris GS115m/LacB greatly improved recombinant protein production relative to the parental strain (P. pastoris GS115/LacB). In the present study, transcriptomes of P. pastoris GS115m/LacB and P. pastoris GS115/LacB grown in YPR medium were analyzed. P. pastoris GS115m/LacB was found to suffer from the mild carbon source starvation. To attenuate the starvation stress, P. pastoris GS115m/LacB attempted to enhance rhamnose metabolism by elevating the transcription levels of rhamnose-utilization genes LRA1-3 and RhaR. The transcription level of LacB under the control of P LRA3 thereby increased, resulting in the improved production of recombinant protein in P. pastoris GS115m/LacB. It was also revealed that P. pastoris GS115m/LacB cells coped with carbon starvation mostly via autophagy.

show abstract

Discovery of a rhamnose utilization pathway and rhamnose-inducible promoters in Pichia pastoris

Cited by 27 publications

References 22 publications

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa

Decreased Rhamnose Metabolic Flux Improved Production of Target Proteins and Cell Flocculation in Pichia pastoris

Decreased expression of LRA4, a key gene involved in rhamnose metabolism, caused up-regulated expression of the genes in this pathway and autophagy in Pichia pastoris

Contact Info

Product

Resources

About