Repeated-batch fermentations of xylose and glucose–xylose mixtures using a respiration-deficient <i>Saccharomyces cerevisiae</i> engineered for xylose metabolism

Kim, Soo Rin,Lee, Ki-Sung,Choi, Jin-Ho,Ha, Suk-Jin,Kweon, Dae-Hyuk,Seo, Jin-Ho,Jin, Yong-Su Elsevier 2010 Journal of biotechnology Vol.150 No.3

<P><B>Abstract</B></P><P>Xylose-fermenting <I>Saccharomyces</I> strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered <I>Saccharomyces cerevisiae</I> strains expressing <I>XYL1</I>, <I>XYL2</I> and <I>XYL3</I> from <I>Pichia stipitis</I>, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered <I>S. cerevisiae</I> to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46gg<SUP>−1</SUP>) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3gL<SUP>−1</SUP>h<SUP>−1</SUP>) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3gL<SUP>−1</SUP>h<SUP>−1</SUP>). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35gg<SUP>−1</SUP>) and productivities (0.52gL<SUP>−1</SUP>h<SUP>−1</SUP>) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30gg<SUP>−1</SUP>) were higher than ethanol yields from xylose as a sole carbon source (0.21gg<SUP>−1</SUP>). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.</P>

Application of a Compatible Xylose Isomerase in Simultaneous Bioconversion of Glucose and Xylose to Ethanol

Chandrakant Priya,Bisaria Virendra S. The Korean Society for Biotechnology and Bioengine 2000 Biotechnology and Bioprocess Engineering Vol.5 No.1

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeast Saccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose and S. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and $30^{\circ}C$. This compatible xylose isomerase from Candida boidinii, having an optimum pH and temperature range of 4.5-5.0 and $30-35^{\circ}C$ respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol by S. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of $42.8\%$.

Microaerobic conversion of xylose to ethanol in recombinant Saccharomyces cerevisiae SX6^MUT expressing cofactor-balanced xylose metabolic enzymes and deficient in ALD6

Jo, S.E.,Seong, Y.J.,Lee, H.S.,Lee, S.M.,Kim, S.J.,Park, K.,Park, Y.C. Elsevier Science Publishers 2016 Journal of biotechnology Vol.227 No.-

<P>Xylose is a major monosugar in cellulosic biomass and should be utilized for cost-effective ethanol production. In this study, xylose-converting ability of recombinant Saccharomyces cerevisiae SX6MUT expressing NADH-preferring xylose reductase mutant (R276H) and other xylose-metabolic enzymes, and deficient in aldehyde dehydrogenase 6 (Ald6p) were characterized at microaerobic conditions using various sugar mixtures. The reduction of air supply from 0.5 vvm to 0.1 vvm increased specific ethanol production rate by 75% and did not affect specific xylose consumption rate. In batch fermentations using various concentrations of xylose (50-104 g/L), higher xylose concentration enhanced xylose consumption rate and ethanol productivity but reduced ethanol yield, owing to the accumulation of xylitol and glycerol from xylose. SX6MUT consumed monosugars in pitch pine hydrolysates and produced 23.1 g/L ethanol from 58.7 g/L sugars with 0.39 g/g ethanol yield, which was 14% higher than the host strain of S. cerevisiae D452-2 without the xylose assimilating enzymes. In conclusion, S. cerevisiae SX6(MUT) was characterized to possess high xylose-consuming ability in microaerobic conditions and a potential for ethanol production from cellulosic biomass. (C) 2016 Elsevier B.V. All rights reserved.</P>

Ethanol Production by In situ Xylose Isomerization Using Recombinant Escherichia coli and Fermentation Using Conventional Saccharomyces cerevisiae

Zhibin Liu,Jinchuan Wu 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.5

Xylose isomerase from Geobacillus kaustophilus HTA426 was functionally expressed in Escherichia coli BL21 (DE3) and the recombinant E. coli cells were used together with conventional Saccharomyces cerevisiae to produce ethanol from xylose by simultaneous xylose isomerisation and fermentation. When recombinant E. coli cells were used as the source of xylose isomerase, a significant amount of ethanol was produced from xylose,whereas the control without recombinant E. coli cells did not produce any detectable amount of ethanol from xylose. Ethanol production was increased by 38% by feeding more recombinant E. coli at 48 h compared to adding recombinant E. coli only in the beginning, resulting in more ethanol production than P. stipitis CBS6054 under the same conditions. The xylitol accumulation by the in situ process was only 57% of that produced by the P. stipitis CBS6054. Xylose isomerase from Geobacillus kaustophilus HTA426 was functionally expressed in Escherichia coli BL21 (DE3) and the recombinant E. coli cells were used together with conventional Saccharomyces cerevisiae to produce ethanol from xylose by simultaneous xylose isomerisation and fermentation. When recombinant E. coli cells were used as the source of xylose isomerase, a significant amount of ethanol was produced from xylose,whereas the control without recombinant E. coli cells did not produce any detectable amount of ethanol from xylose. Ethanol production was increased by 38% by feeding more recombinant E. coli at 48 h compared to adding recombinant E. coli only in the beginning, resulting in more ethanol production than P. stipitis CBS6054 under the same conditions. The xylitol accumulation by the in situ process was only 57% of that produced by the P. stipitis CBS6054.

Construction of an efficient xylose-fermenting diploid Saccharomyces cerevisiae strain through mating of two engineered haploid strains capable of xylose assimilation

Kim, S.R.,Lee, K.S.,Kong, I.I.,Lesmana, A.,Lee, W.H.,Seo, J.H.,Kweon, D.H.,Jin, Y.S. Elsevier Science Publishers 2013 Journal of biotechnology Vol.164 No.1

Saccharomyces cerevisiae can be engineered for xylose fermentation through introduction of wild type or mutant genes (XYL1/XYL1 (R276H), XYL2, and XYL3) coding for xylose metabolic enzymes from Scheffersomyces stipitis. The resulting engineered strains, however, often yielded undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. In this study, we performed the mating of two engineered strains that exhibit suboptimal xylose-fermenting phenotypes in order to develop an improved xylose-fermenting diploid strain. Specifically, we obtained two engineered haploid strains (YSX3 and SX3). The YSX3 strain consumed xylose rapidly and produced a lot of xylitol. On the contrary, the SX3 strain consumed xylose slowly with little xylitol production. After converting the mating type of SX3 from alpha to a, the resulting strain (SX3-2) was mated with YSX3 to construct a heterozygous diploid strain (KSM). The KSM strain assimilated xylose (0.25gxyloseh<SUP>-1</SUP>gcells<SUP>-1</SUP>) as fast as YSX3 and accumulated a small amount of xylitol (0.03ggxylose<SUP>-1</SUP>) as low as SX3, resulting in an improved ethanol yield (0.27ggxylose<SUP>-1</SUP>). We found that the improvement in xylose fermentation by the KSM strain was not because of heterozygosity or genome duplication but because of the complementation of the two xylose-metabolic pathways. This result suggested that mating of suboptimal haploid strains is a promising strategy to develop engineered yeast strains with improved xylose fermenting capability.

대장균 xylA 프로모터를 이용한 xylose 유도성 발현벡터의 구축

김현호,소재현,이인구 한국응용생명화학회 2010 Journal of Applied Biological Chemistry (J. Appl. Vol.53 No.1

xylA promoter is a major promoter in xylose operon of Escherichia coli. xylA promoter is sufficient as the promoter for the construction of new expression vector because this promoter was tightly controlled and induced by the addition of xylose. For the construction of xylose-inducible expres-sion vector, 600 bp of xylA promoter was ligated between AatII and HindIII of pUC18, named pXA600. In order to investigate the effect of XylR protein encoded by xylR gene on the xylA pro-moter, 1,988 bp of xylR gene including its promoter was ligated into downstream of multiple clon-ing site to the opposite direction of xylA promoter in pXA600, named pXAR600. For the measurement of expression level, 3,048 bp of lacZ structural gene was fused into xylA promoter in both plasmids pXA600 and pXAR600 as a reporter gene, named pXA600-lacZ and pXAR600-lacZ, respectively. The β-galactosidase activity of pXA600-lacZ and pXAR600-lacZ in E. coli JM109 was determined to be 1,641 and 2,304 unit by the induction with xylose in LB medium, respectively. The β-galactosidase activity of pXAR600-lacZ/JM109 was about 1.4 times higher by the induction with xylose than that of pXA600-lacZ/JM109. The β-galactosidase activity of pXA600-lacZ and pXAR600-lacZ in E.coli JM109 showed 6,282 and 9,320 unit by the induction with xylose in DM minimal medium, respectively. A regulator, xylR protein works as an activator for the gene expres-sion by the addition of xylose in the xylose-inducible vectors because the level of gene expression in pXA600 is increased by the insertion of xylR gene into the same vector. The xynA gene of Strep-tomyces thermocyaneoviolaceus cloned in pXA600 and pXAR600 was successfully expressed in E. coli BLR(DE3). As a result, plasmids pXA600 and pXAR600 using xylA promoter are sufficient as new expression system to produce a foreign protein in E. coli. xylA 프로모터는 대장균의 xylose 대사에 관여하는 xylose 오페론 상의 중요한 프로모터이다. 이 프로모터는 xylose에 의해 강하게 조절을 받는다고 알려져 있다. 이러한 특징은 새로운 발현 백터를 구축하는데 충분한 조건을 갖추고 있다고 생각된다. 본 연구에서는 이러한 xylose에 의해 유도 되는 발현벡터를 구축하기 위하여 600 bp의 xylA 프로모터를 증폭하여 pUC18의 AatII와 HindIII 사이에 삽입하여 pXA600을 구축하였다. 또한 조절단백질인 XylR의 영향을 조사하기 위하여 xylR 유전자를 삽입하여 pXAR600을 구축하였다. 발현의 강도를 측정하기 위하여 3,048 bp의 lacZ유전자를 xylA 프로모터의 하류에 연결하여 pXA600-lacZ와 pXAR600-lacZ를 구축하고 대장균 JM109에 형질전환시켰다. 구축된 pXA600-lacZ와 pXAR600-lacZ는 LB 배지에서 배양하였을 때 xylose 유도하에서 각각 1,641 unit와 2,304 unit의 β-galactosidase 활성을 보였으며, DM 배지상에 서 배양했을 때 xylose 유도 시 각각 6,282 unit와 9,320 unit의 β-galactosidase 활성을 보였다. 또한 왜래 유전자의 발현 가능성을 확인하기 위하여 S. thermocyaneoviolaceus의 내열성 xylanase를 코딩하는 xynA 유전자를 실제로 구축된 pXA600과 pXAR600에서 발현을 확인하여 pXA600 및 pXAR600이 새로운 xylose 유도성 발현벡터로서의 사용 가능성을 확인하였다.

Xylitol 생산에 최적화된 xylose reductase (GRE3)의 분비발현 시스템

정회명(Hoe-Myung Jung),김재운(Jae-Woon Kim),김연희(Yeon-Hee Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.12

Xylitol은 식품 및 의료산업에서 이용가치가 높은 물질로, lignocellulosic biomass인 xylose의 환원으로부터 생산되며, 대부분 유전적으로 안전한 Saccharomyces cerevisiae 균주를 사용하여 생산되고 있다. 따라서 본 연구에서는 S. cerevisiae에서 xylitol을 효율적으로 생산하기 위해 xylose reductase를 code하는 GRE3 (YHT104W)유전자의 발현시스템을 구축하여, xylose reductase의 분비생산 및 xylitol 생산성을 조사하고자 하였다. 먼저 GRE3 유전자의 발현에 적합한 promoter의 선별을 위해 GAL10 promoter와 ADH1 promoter 하류에 각각 mating factor α (MFα) signal sequence와 GRE3 유전자를 가진 pGMF-GRE3와 pAMF-GRE3 plasmid를 구축하였다. 각각의 plasmid는 S. cerevisiae SEY2102△trp1균주에 형질전환되었고, SEY2102△trp1/pGMF-GRE3와 SEY2102△trp1/pAMF-GRE3 형질전환주가 선별되었다. 그 중 SEY2102△trp1/pGMF-GRE3 균주에서 NADPH를 cofactor로 사용했을 때 0.34 unit/mg-protein의 xylose reductase 활성(total activity)을 보였고, ADH1 promoter를 가진 SEY2102△trp1/pAMF-GRE3 균주에 비해 1.5배 높은 활성증가를 확인 할 수 있었다. 또한 두 균주에서 모두 91%의 분비효율을 보여 대부분의 재조합 xylose reductase가 세포 밖으로 효율적으로 발현 분비되었음을 알 수 있었다. SEY2102△trp1/pGMF-GRE3 균주를 사용한 baffled flask 배양에서 xylitol 생산량을 조사해 본 결과, 20 g/l의 xylose로부터 12.1 g/l의 xylitol을 생산하였고, 소모된 xylose의 약 83%정도가 xylitol로 환원되었음을 알 수 있었다. Xylitol is widely used in the food and medical industry. It is produced by the reduction of xylose (lignocellulosic biomass) in the Saccharomyces cerevisiae strain, which is considered genetically safe. In this study, the expression system of the GRE3 (YHR104W) gene that encodes xylose reductase was constructed to efficiently produce xylitol in the S. cerevisiae strain, and the secretory production of xylose reductase was investigated. To select a suitable promoter for the expression of the GRE3 gene, pGMF-GRE3 and pAMF-GRE3 plasmid with GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor α (MFα) signal sequence was also connected to each promoter for secretory production. Each plasmid was transformed into S. cerevisiae SEY2102△trp1, and SEY2102△ trp1/pGMF- GRE3 and SEY2102△trp1/pAMF-GRE3 transformants were selected. In the SEY2102△ trp1/pGMF-GRE3 strain, the total activity of xylose reductase reached 0.34 unit/mg-protein when NADPH was used as a cofactor; this activity was 1.5 fold higher than that in SEY2102△trp1/pAMF-GRE3 with ADH1 as the promoter. The secretion efficiency was 91% in both strains, indicating that most of the recombinant xylose reductase was efficiently secreted in the extracellular fraction. In a baffled flask culture of the SEY2102△trp1/pGMF-GRE3 strain, 12.1 g/l of xylitol was produced from 20 g/l of xylose, and ~83% of the consumed xylose was reduced to xylitol.

Enhanced production of 2,3-butanediol from xylose by combinatorial engineering of xylose metabolic pathway and cofactor regeneration in pyruvate decarboxylase-deficient <i>Saccharomyces cerevisiae</i>

Kim, Soo-Jung,Sim, Hee-Jin,Kim, Jin-Woo,Lee, Ye-Gi,Park, Yong-Cheol,Seo, Jin-Ho Elsevier Applied Science 2017 Bioresource technology Vol.245 No.2

<P><B>Abstract</B></P> <P>The aim of this study was to produce 2,3-butanediol (2,3-BDO) from xylose efficiently by modulation of the xylose metabolic pathway in engineered <I>Saccharomyces cerevisiae</I>. Expression of the <I>Scheffersomyces stipitis</I> transaldolase and NADH-preferring xylose reductase in <I>S. cerevisiae</I> improved xylose consumption rate by a 2.1-fold and 2,3-BDO productivity by a 1.8-fold. Expression of the <I>Lactococcus lactis noxE</I> gene encoding NADH oxidase also increased 2,3-BDO yield by decreasing glycerol accumulation. Additionally, the disadvantage of C<SUB>2</SUB>-dependent growth of pyruvate decarboxylase-deficient (Pdc<SUP>−</SUP>) <I>S. cerevisiae</I> was overcome by expression of the <I>Candida tropicalis PDC1</I> gene. A fed-batch fermentation of the BD5X-TXmNP strain resulted in 96.8g/L 2,3-BDO and 0.58g/L-h productivity from xylose, which were 15.6- and 2-fold increases compared with the corresponding values of the BD5X strain. It was concluded that facilitation of the xylose metabolic pathway, oxidation of NADH and relief of C<SUB>2</SUB>-dependency synergistically triggered 2,3-BDO production from xylose in Pdc<SUP>−</SUP> <I>S. cerevisiae</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Overexpression of <I>TAL1</I> improved 2,3-BDO productivity from xylose. </LI> <LI> Expression of mutant XR contributed to enhancing xylose consumption rate. </LI> <LI> Expression of <I>noxE</I> increased 2,3-BDO yield from xylose by reducing glycerol production. </LI> <LI> Introduction of <I>C. tropicalis PDC1</I> allowed to escape from C<SUB>2</SUB>-auxotrophy. </LI> <LI> Optimal fed-batch fermentation resulted in 96.8g/L of 2,3-BDO from xylose. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>2,3-Butanediol biosynthetic pathway from xylose and glucose in recombinant C<SUB>2</SUB>-independent and Pdc-deficient <I>S. cerevisiae;</I> The superscripts in the enzyme names present as follows: <I>Bs</I>, <I>Bacillus subtilis; Ct</I>, <I>Candida tropicalis; Ll</I>, <I>Lactococcus lactis; Ss, Scheffersomyces stipitis</I>; The names of enzymes are abbreviated as follow: XR, xylose reductase; XDH, xylitol dehydrogenase; XK, xylulose kinase; TAL1, transaldolase; ALS, α-acetolactate synthase; ALDC, α-acetolactate decarboxylase; BDH, 2,3-BDO dehydrogenase; PDC, pyruvate decarboxylase; NoxE, NADH oxidase.</P> <P>[DISPLAY OMISSION]</P>

Construction of efficient xylose-fermenting <i>Saccharomyces cerevisiae</i> through a synthetic isozyme system of xylose reductase from <i>Scheffersomyces stipitis</i>

Jo, Jung-Hyun,Park, Yong-Cheol,Jin, Yong-Su,Seo, Jin-Ho Elsevier 2017 Bioresource technology Vol.241 No.-

<P><B>Abstract</B></P> <P>Engineered <I>Saccharomyces cerevisiae</I> has been used for ethanol production from xylose, the abundant sugar in lignocellulosic hydrolyzates. Development of engineered <I>S. cerevisiae</I> able to utilize xylose effectively is crucial for economical and sustainable production of fuels. To this end, the xylose-metabolic genes (<I>XYL1</I>, <I>XYL2</I> and <I>XYL3</I>) from <I>Scheffersomyces stipitis</I> have been introduced into <I>S. cerevisiae.</I> The resulting engineered <I>S. cerevisiae</I> strains, however, often exhibit undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. This work was undertaken to construct an improved xylose-fermenting strain by developing a synthetic isozyme system of xylose reductase (XR). The DXS strain having both wild XR and mutant XR showed low xylitol accumulation and fast xylose consumption compared to the engineered strains expressing only one type of XRs, resulting in improved ethanol yield and productivity. These results suggest that the introduction of the XR-based synthetic isozyme system is a promising strategy to develop efficient xylose-fermenting strains.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The engineered <I>S. cerevisiae</I> strain having both wild XR and mutant XR was constructed. </LI> <LI> The XR based isozyme was used to extend cofactor availability. </LI> <LI> Coexpressing of wild XR and mutant XR improved the bioethanol productivity and yield. </LI> <LI> The ethanol productivity of 1.85g/L·h and yield of 0.427g/g were obtained. </LI> </UL> </P>

Improvement of Xylose Utilization in metabolically-engineered Corynebacterium glutamicum

박재현,강석원,우한민 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the D-xylose, which is second most abundant sugar-derived hemicellulose, for cell growth. Therefore, we introduced xylose isomerase pathway to xylose-negative C. glutamicum and overexpression of the pentose phosphate pathway genes and a heterologous phosphoketolase (PHK) pathway gene for xylose utilization. Overexpression of the genes encoding for tal, gnd, or xpkA improved the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. The engineered strains, including overexpression of the tal, gnd, or xpkA gene alone and co-overexpression of the tal and xpkA genes, with varying xylose concentrations using a BioLector. Interestingly, xylose-utilizing C. glutamicum strains showed increased specific growth rates at higher concentrations of xylose. This study could be useful for amino acid and other value-added chemical production from xylose as the sole carbon source.