Cloning and expression of Candida guilliermondii xylose reductase gene ( xyl1 ) in Pichia pastoris

1998 ◽  
Vol 49 (4) ◽  
pp. 399-404 ◽  
Author(s):  
C. Handumrongkul ◽  
D.-P. Ma ◽  
J. L. Silva
Keyword(s):  
Pichia Pastoris ◽  
Xylose Reductase ◽  
Candida Guilliermondii ◽  
Cloning And Expression ◽  
Reductase Gene

scholarly journals Production of bio-xylitol from d-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Tai Man Louie ◽  
Kailin Louie ◽  
Samuel DenHartog ◽  
Sridhar Gopishetty ◽  
Mani Subramanian ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Electron Donor ◽  
Low Cost ◽  
Standard Procedure ◽  
Xylose Reductase ◽  
Animal Nutrition ◽  
Whole Cells ◽  
Reductase Gene ◽  
Biocatalytic Reaction ◽  
Ketone Reductases

Abstract Background Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from d-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from d-xylose. Results Pichia pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform d-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield. Conclusions We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform d-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of d-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

scholarly journals Production of bio-xylitol from D-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

2021 ◽  
Author(s):  
Michael Louie ◽  
Kailin Louie ◽  
Samuel DenHartog ◽  
Sridhar Gopishetty ◽  
Mani Subramanian ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Electron Donor ◽  
Low Cost ◽  
Standard Procedure ◽  
Xylose Reductase ◽  
Animal Nutrition ◽  
Whole Cells ◽  
Reductase Gene ◽  
Biocatalytic Reaction ◽  
Ketone Reductases

Abstract Background: Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from D-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from D-xylose.Results: P. pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform D-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield.Conclusions: We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform D-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of D-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

scholarly journals Production of bio-xylitol from D-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

2020 ◽  
Author(s):  
Michael Louie ◽  
Kailin Louie ◽  
Samuel DenHartog ◽  
Sridhar Gopishetty ◽  
Mark Arnold ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Electron Donor ◽  
Low Cost ◽  
Standard Procedure ◽  
Xylose Reductase ◽  
Animal Nutrition ◽  
Whole Cells ◽  
Reductase Gene ◽  
Biocatalytic Reaction ◽  
Ketone Reductases

Abstract Background: Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from D-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from D-xylose.Results: P. pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform D-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield.Conclusions: We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform D-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of D-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

scholarly journals Production of bio-xylitol from D-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

2020 ◽  
Author(s):  
Michael Louie ◽  
Kailin Louie ◽  
Samuel DenHartog ◽  
Sridhar Gopishetty ◽  
Mani Subramanian ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Electron Donor ◽  
Low Cost ◽  
Standard Procedure ◽  
Xylose Reductase ◽  
Whole Cells ◽  
Reductase Gene ◽  
Recombinant Pichia Pastoris ◽  
Biocatalytic Reaction ◽  
Ketone Reductases

Abstract Background: Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from D-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from D-xylose. Results: P. pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform D-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield. Conclusions: We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform D-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P) + , and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of D-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

CLONING AND EXPRESSION OF SILURUS.MERIDIUNLIS OBESE GENE IN PICHIA. PASTORIS

2008 ◽  
Vol 32 (1) ◽  
pp. 112-115
Author(s):  
Fen NIE
Keyword(s):  
Pichia Pastoris ◽  
Cloning And Expression ◽  
I Gene

Cloning and Expression of a Novel Xylanase Gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris

2012 ◽  
Vol 167 (8) ◽  
pp. 2198-2211 ◽  
Author(s):  
Huimin Zhang ◽  
Minchen Wu ◽  
Jianfang Li ◽  
Shujuan Gao ◽  
Yanjun Yang
Keyword(s):  
Pichia Pastoris ◽  
Cloning And Expression ◽  
Xylanase Gene ◽  
Aspergillus Usamii

scholarly journals Obtaining partial purified xylose reductase from Candida guilliermondii

2009 ◽  
Vol 40 (3) ◽  
pp. 631-635 ◽  
Author(s):  
Ester Junko Tomotani ◽  
Priscila Vaz de Arruda ◽  
Michele Vitolo ◽  
Maria das Graças de Almeida Felipe
Keyword(s):  
Xylose Reductase ◽  
Candida Guilliermondii

Xylose Reductase Activity of Candida guilliermondii During Xylitol Production by Fed-Batch Fermentation

2002 ◽  
pp. 875-883
Author(s):  
Denise C. G. A. Rodrigues ◽  
Silvio S. Da Silva ◽  
J. B. Almeida E Silva ◽  
Michele Vitolo
Keyword(s):  
Batch Fermentation ◽  
Reductase Activity ◽  
Xylose Reductase ◽  
Candida Guilliermondii ◽  
Xylitol Production ◽  
Fed Batch ◽  
Fed Batch Fermentation
Sign in / Sign up

Export Citation Format

Share Document