Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol

Flocculent yeast Saccharomyces cerevisiae YF234 (MATa ura3–52 trp1Δ2 his ade 2–1 can1–100 sta1 FLO8) cells overexpressing glyoxalase I and having strong flocculation ability were permeabilized with isopropyl alcohol and ethanol under various conditions. The treatment with 40% isopropyl alcohol significantly improves the initial reaction rates of recombinant flocculent yeast cells. Moreover, the reactivity of permeabilized flocculent yeast cells was similar to that of dispersed cells with EDTA. On the other hand, the flocculation ability of yeast cells was not affected by the treatment with alcohol solutions of various concentrations and treatment time length. Therefore, the recombinant flocculent yeast cells permeabilized with alcohol are very effective whole cell biocatalysts.     

Efficient anaerobic whole cell stereoselective bioreduction with recombinant Saccharomyces cerevisiae

In this study we investigate the NADPH-dependent stereoselective reduction of the bicyclic diketone bicyclo[2.2.2]octane-2,6-dione (BCO2,6D) to the chiral ketoalcohol (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one (BCO2one6ol). Our aim was to develop a whole cell batch process for reduction of carbonyl substrates with (i) a high cosubstrate yield (formed product/consumed cosubstrate) and (ii) a high conversion rate under anaerobic conditions with Saccharomyces cerevisiae as biocatalyst and glucose as cosubstrate. Five open reading frames (ORFs), YMR226c, YDR368w, YOR120w, YGL157w, and YGL039w, encoding reductases involved in the conversion of BCO2,6D were identified using cell-free extract from strains belonging to the ExClone collection (yeast ORF expression clones; ResGen, Invitrogen Corp., UK). We report the one-step purification and characterization of three major BCO2,6D reductases, YMR226cp, YDR368wp (YPR1p), and YOR120wp (GCY1p). The reductases were overexpressed under a strong constitutive promoter and the impact on cosubstrate yield, conversion time, glucose consumption rate, and reduction rate was investigated when reductases were overexpressed either alone or in combination with low phosphoglucose isomerase activity (encoded by YBR196c). Combining overexpression of BCO2,6D reductase with reduced glycolytic rate (low phosphoglucose isomerase activity) offers a fast whole cell stereoselective bioreduction system useful for facilitated anaerobic batch conversions.     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.     

酿酒酵母S.cerevisiae高密度培养条件优化研究

考察了培养基组成和培养条件对酿酒酵母Saccharomyces cerevisiae发酵的影响。以TB培养基为初始培养基,通过正交实验设计优化培养基组成,确定了影响酵母细胞产量最主要的因素是葡萄糖,最适培养基组成为:酪蛋白胨15 g/L,酵母粉25 g/L,葡萄糖30 g/L,KH2PO42.4g/L,K2HPO4.3H2O 16.34 g/L。并确定了最佳培养条件:温度30℃,转速150 r/min。采用优化培养基及培养条件下进行发酵,菌液最高OD600值和细胞密度分别达15.82和2.03×108/mL,比优化前分别提高24.2%和22.0%。     

Stability studies of recombinant Saccharomyces cerevisiae in the presence of varying selection pressure

A recombinant yeast plasmid carrying the Ieu2 gene for auxotrophic complementation and a reporter gene for beta-galactosidase under the control of Gal10 promoter was studied in Saccharomyces cerevisiae. Growth, product formation, and plasmid stability were studied in defined, semi-defined, and complex media. The biomass concentration and specific activity were higher in complex medium than in defined medium, which was selective for the growth of plasmid-containing cells, leading to a 10-fold increase in volumetric activity. However, plasmid instability was very high in complex media with 50% plasmid-free cells emerging in the culture within 75 h of cultivation. In order to control instability, the growth rates of the plasmid-containing and plasmid-free cells were determined in semi-defined media, which consisted of defined medium supplemented with different concentrations of yeast extract. Below a critical concentration of yeast extract (0.05 g/L), the plasmid-containing cells had a growth rate advantage over the plasmid-free cells. This was possibly because, at this concentration of yeast extract, the availability of leucine became the rate-determining factor in the specific growth rate of plasmid-free cells. A feeding strategy was designed which maintained a low concentration of the residual yeast extract in the medium and thus continuously provided the plasmid-containing cells with a competitive advantage over the plasmid-free cells. This resulted in high stability as well as high cell density under non-selective conditions, which led to a 10-fold increase in the volumetric activity compared to that achieved in defined selective media. A simple mathematical model was formulated to verify the experimental data. The important state variables and process parameters, i.e., biomass concentration, beta-galactosidase expression, sucrose consumption, yeast extract consumption, and specific growth rates of the two cell populations, were evaluated. These variables and parameters along with the differential equations based on material balances as well as the experimental results obtained were used in a mathematical model for the fed-batch cultivation. These correctly verified the experimental data and clearly illustrated the concept behind the success of the fed-batch strategy under yeast extract starvation.     

PAG-OA衍生物I5对酿酒酵母转化合成ATP的影响

一种新型促渗透剂PAG-OA（聚氧烯油酸二醇）I5,甲苯、气流干燥及吐温100等,对酿酒酵母进行细胞渗透性增强处理,考察了酿酒酵母的促渗透性对三磷酸腺苷生产的影响。结果表明,与其他促渗透方式相比,I5对酿酒酵母三磷酸腺苷的产量有很大的提高。加入0．022mol／L腺苷,三磷酸腺苷得率为0．038mol／L,转化率98％;三磷酸腺苷合成时间缩短为1．5h。经促渗透化处理的酿酒酵母细胞能很好的释放胞内代谢的极性物质;其三磷酸腺苷生产活性大幅度提高。     

重组大肠杆菌全细胞催化合成莱鲍迪苷D

莱鲍迪苷D(Rebaudioside D,RD)是一种稀有具有高甜度的甜菊糖苷类化合物。本文实现了重组大肠杆菌全细胞催化莱鲍迪苷A(Rebaudioside A,RA)合成RD。以水稻c DNA为模板,扩增得到葡萄糖基转移酶基因eugt11,构建了重组菌株E.coli BL21(p ETDuet-eugt11),并成功表达了重组蛋白6His-EUGT11。通过Ni柱亲和层析纯化并在体外酶催化反应表征了其催化活性。将重组菌BL21(p ETDuet-eugt11)应用于催化合成RD研究。探讨了反应体系pH、温度、柠檬酸钠浓度、菌体密度、二价金属离子、二甲苯体积分数、UDPG添加浓度对反应效率的影响。单因素考察结果显示,在菌体密度0.16 g湿细胞/m L反应液,底物RA浓度为1.0 mmol/L,pH 8.0,60 mmol/L柠檬酸钠,1%二甲苯,0.1 mmol/L Zn Cl2,12.0 mmol/L UDPG,反应温度42℃,反应时间24 h的条件下,RD产量为123.6 mg/L(约0.1 mmol/L)。     

Production of wild-type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)(2) and (WP)(4), respectively. Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg(-1) with activity per protein broth (specific activity) of 266 Umg(-1), whereas cutinase-(WP)(2) was secreted with a specific cell activity of 2.1 Umg(-1) with a specific activity of 200 Umg(-1), and cutinase-(WP)(4) with a specific cell activity of 0.7 Umg(-1) with a specific activity of 15 Umg(-1). The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture).     

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.     

Optimization of 1,2,4‐butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance

1,2,4‐Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4‐butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2‐ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2‐keto‐3‐deoxy‐xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2‐ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.     

EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker‐free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone‐MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113‐7D) and a diploid industrial strain (Ethanol Red) for production of 3‐hydroxypropionic acid, where we tested three different acetyl‐CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone‐MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.     

Green synthesis of enantiopure (S)‐1‐(benzofuran‐2‐yl)ethanol by whole‐cell biocatalyst

Optically active aromatic alcohols are valuable chiral building blocks of many natural products and chiral drugs. Lactobacillus paracasei BD87E6, which was isolated from a cereal‐based fermented beverage, was shown as a biocatalyst for the bioreduction of 1‐(benzofuran‐2‐yl) ethanone to (S)‐1‐(benzofuran‐2‐yl) ethanol with highly stereoselectivity. The bioreduction conditions were optimized using L. paracasei BD87E6 to obtain high enantiomeric excess (ee) and conversion. After optimization of the bioreduction conditions, it was shown that the bioreduction of 1‐(benzofuran‐2‐yl)ethanone was performed in mild reaction conditions. The asymmetric bioreduction of the 1‐(benzofuran‐2‐yl)ethanone had reached 92% yield with ee of higher than 99.9% at 6.73 g of substrate. Our study gave the first example for enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol by a biological green method. This process is also scalable and has potential in application. In this study, a basic and novel whole‐cell mediated biocatalytic method was performed for the enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol in the aqueous medium, which empowered the synthesis of a precious chiral intermediary process to be converted into a sophisticated molecule for drug production.