Xuewu Guo

Production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82 with pH control and DL-dithiothreitol addition

Xylose, the second most abundant sugar in lignocellulosic materials, is not efficiently utilized in current lignocellulose biotransformation processes, such as cellulosic ethanol production. The bioconversion of xylose to value-added products, such as pullulan, is an alternative strategy for efficient lignocellulose biotransformation. This paper reports the production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82. The effects of DL-dithiothreitol (DTT) and pH on pullulan production from xylose were also intensively investigated. A maximal increase of 17.55% of pullulan production was observed in flasks added with 1.0 mM DTT. Batch fermentations with controlled pH were also conducted, and the optimal pH for cell growth and pullulan synthesis was 3.0 and 5.0, respectively. Based on these findings, two-stage pH control fermentations were performed, in which the pH of the medium was first adjusted to 3.0 for cell growth, and then changed to 5.0 for pullulan synthesis. However, the earlier the pH was changed to 5.0, the more pullulan was produced. Fermentation with controlled pH of 5.0 acquired the highest pullulan production. Under the optimized conditions (with the addition of 1.0 mM DTT and controlled pH of 5.0), the maximal pullulan production obtained from xylose was 17.63 g/L. A. pullulans AY82 also readily fermented hemicellulose hydrolysate under these optimized conditions, but with lower pullulan production (12.65 g/L). Fourier transform infrared spectroscopy and high-performance liquid chromatography showed that the structure of the pullulan obtained in this study was identical to that of the pullulan standard.

Optimization of 2,3‐butanediol production by Enterobacter cloacae in simultaneous saccharification and fermentation of corncob residue

Corncob residue, a waste in xylose or xylitol production, was utilized to produce 2,3‐butanediol (2,3‐BD) via simultaneous saccharification and fermentation (SSF). This study developed the optimal conditions for production of 2,3‐BD by using a heat‐resistant strain, Enterobacter cloacae UV4, to perform SSF of the corncob residue. Urea, lactic acid, sodium citrate, and MgSO4, selected by the Plackett–Burman experiment, were determined to be significant independent variables to conduct the response surface experiment. With the optimized medium, a total production of 28.923 g/L for 2,3‐BD and acetoin (BA) was obtained at 60 H. Furthermore, 43.162 g/L of BA production and 0.553 g/L/H of productivity were obtained by fed‐batch SSF, which was 0.424 g diol/g consumed corncob residue. The results suggest that the waste corncob residue could be used as an available substrate for the production of 2,3‐BD by E. cloacae UV4, as well as a potential resource to improve the economics of microbial compound production.

Efficient utilization of hemicellulose and cellulose in alkali liquor-pretreated corncob for bioethanol production at high solid loading by Spathaspora passalidarum U1-58

The bioethanol fermentation of pretreated corncob was investigated using Spathaspora passalidarum U1-58, which simultaneously utilizes glucose and xylose for high-efficiency ethanol production. Two approaches, namely, separate hydrolysis and co-fermentation (SHCF) and simultaneous saccharification and co-fermentation (SSCF), were optimized to test the ethanol fermentation potential of U1-58. The highest ethanol titer of 42.46g/L and yield of 72.12% were acquired in SHCF, whereas 53.24g/L ethanol and yield of 75.35% were obtained in SSCF at solid-to-liquid ratio of 1:5 (w/v). Approximately 86.20% of cellulose and 82.99% of hemicellulose were consumed in SSCF after 96h, and at least 10.49g/L ethanol was produced from hemicellulose, which corresponded to 37.59% of the theoretical yield. Compared with the published cellulosic ethanol fermentation cases, the present work presented high ethanol titer and yield, and cellulose and hemicellulose could be efficiently utilized for ethanol production.

A rapid and efficient one-step site-directed deletion, insertion, and substitution mutagenesis protocol

A rapid and efficient site-directed mutagenesis (SDM) protocol based on two separate polymerase chain reaction (PCR) amplifications and homologous recombination in Escherichia coli is described. This protocol can introduce deletions, substitutions, and insertions into any amplifiable site of the target genes by ligating two amplified DNA fragments into vectors. Compared with previously reported PCR-based SDM methods, our protocol significantly prevents primer dimerization even though partially complementary primers were used for PCR. The genome with the target gene was used directly as template, and DpnI was unnecessary. All of the procedures were performed within 24 h. The mutation frequencies of deletion, substitution, and insertion of the PEP4 (encode proteinase A) gene of Saccharomyces cerevisiae were nearly 100% using this new method. Thus, this method can potentially facilitate high-throughput genetic engineering and structure-function analyses and is useful for molecular biological research.

Reduction of biogenic amines production by eliminating the PEP4 gene in Saccharomyces cerevisiae during fermentation of Chinese rice wine

Biogenic amines in Chinese rice wine have a potential threat of toxicity to human health. In this study, PEP4 gene in Saccharomyces cerevisiae was knocked out in order to evaluate its effect on biogenic amines production; the enzyme encodes proteinase A (PrA), an enzyme that is responsible for the production of free amino acids. It was found that compared to the wild type strain, the PrA activity and amino acid concentration decreased significantly, and the production of biogenic amines in this knockout strain decreased by 25.5%, from 180.1mg/L to 134.2mg/L. Especially, tyramine, cadaverine and histamine concentrations were also decreased by 57.5%, 24.6% and 54.3%, respectively. The main reason for the decrease of biogenic amines may be due to the low concentration of free amino acids. Our results provide a new strategy to minimize the biogenic amine production during fermentation of Chinese rice wine.

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY-5 hybrid.

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.

Improved ethyl caproate production of Chinese liquor yeast by overexpressing fatty acid synthesis genes with OPI1 deletion

During yeast fermentation, ethyl esters play a key role in the development of the flavor profiles of Chinese liquor. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is the characteristic flavor of strong aromatic liquor, which is the best selling liquor in China. In the traditional fermentation process, ethyl caproate is mainly produced at the later fermentation stage by aroma-producing yeast, bacteria, and mold in a mud pit instead of Saccharomyces cerevisiae at the expense of grains and fermentation time. To improve the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae) with less food consumption and shorter fermentation time, we constructed three recombinant strains, namely, α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 by overexpressing acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1), and fatty acid synthase 2 (FAS2) with OPI1 (an inositol/choline-mediated negative regulatory gene) deletion, respectively. In the liquid fermentation of corn hydrolysate, the contents of ethyl caproate produced by α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 increased by 0.40-, 1.75-, and 0.31-fold, correspondingly, compared with the initial strain α5. The contents of other fatty acid ethyl esters (FAEEs) (C8:0, C10:0, C12:0) also increased. In comparison, the content of FAEEs produced by α5-FAS1ΔOPI1 significantly improved. Meanwhile, the contents of acetyl-CoA and ethyl acetate were enhanced by α5-FAS1ΔOPI1. Overall, this study offers a promising platform for the development of pure yeast culture fermentation of Chinese strong aromatic liquor without the use of a mud pit.

A genetic transformation protocol for the xylose-fermenting yeast Spathaspora passalidarum

Spathaspora passalidarum, a recently isolated native xylose‐fermenting yeast, can ferment xylose faster than glucose, and can co‐ferment glucose, xylose, and cellobiose. Studies on S. passalidarum have been reported in terms of fermentation conditions. However, to the best of our knowledge, the molecular modification of S. passalidarum has not been achieved. The lack of successful transformants may be attributed to the absence of an effective transformation method. In the current work, a genetic transformation protocol for S. passalidarum was established. Spathaspora passalidarum was transformed via the lithium acetate method with a plasmid containing the KmR as a selectable marker and the Saccharomyces cerevisiae 2 μ replicon as a replication origin. Several important parameters affecting transformation efficiency were optimized; these parameters included the strain growth period, heat shock time, lithium acetate concentration, and the amount of plasmid DNA. The transformation efficiency reached 170 ± 30 transformants/μg plasmid DNA with the optimized method. This novel transformation system will allow for the genetic analysis and metabolic engineering of S. passalidarum for industrial bioethanol production.

Saccharomyces cerevisiae proteinase A excretion and wine making

Proteinase A (PrA), the major protease in Saccharomyces cerevisiae, plays an essential role in zymogen activation, sporulation, and other physiological processes in vivo. The extracellular secretion of PrA often occurs during alcoholic fermentation, especially in the later stages when the yeast cells are under stress conditions, and affects the quality and safety of fermented products. Thus, the mechanism underlying PrA excretion must be explored to improve the quality and safety of fermented products. This paper briefly introduces the structure and physiological function of PrA. Two transport routes of PrA, namely, the Golgi-to-vacuole pathway and the constitutive Golgi-to-plasma membrane pathway, are also discussed. Moreover, the research history and developments on the mechanism of extracellular PrA secretion are described. In addition, it is briefly discussed that calcium homeostasis plays an important role in the secretory pathway of proteins, implying that the regulation of PrA delivery to the plasma membrane requires the involvement of calcium ion. Finally, this review focuses on the effects of PrA excretion on wine making (including Chinese rice wine, grape wine, and beer brewage) and presents strategies to control PrA excretion.

Decreased proteinase A excretion by strengthening its vacuolar sorting and weakening its constitutive secretion in Saccharomyces cerevisiae

Proteinase A (PrA), encoded by PEP4 gene, is detrimental to beer foam stability. There are two transport pathways for the new synthesized PrA in yeast, sorting to the vacuole normally, or excreting out of the cells under stress conditions. They were designated as the Golgi-to-vacuole pathway and the constitutive secretory pathway, respectively. To reduce PrA excretion in some new way instead of its coding gene deletion, which had a negative effect on cell metabolism and beer fermentation, we modified the PrA transport based on these above two pathways. In the Golgi-to-vacuole pathway, after the verification that Vps10p is the dominant sorting receptor for PrA Golgi-to-vacuolar transportation by VPS10 deletion, VPS10 was then overexpressed. Furthermore, SEC5, encoding exocyst complexes’ central subunit (Sec5p) in the constitutive secretory pathway, was deleted. The results show that PrA activity in the broth fermented with WGV10 (VPS10 overexpressing strain) and W∆SEC5 (SEC5 deletion strain) was lowered by 76.96 and 32.39%, compared with the parental strain W303-1A, at the end of main fermentation. There are negligible changes in fermentation performance between W∆SEC5 and W303-1A, whereas, surprisingly, WGV10 had a significantly improved fermentation performance compared with W303-1A. WGV10 has an increased growth rate, resulting in higher biomass and faster fermentation speed; finally, wort fermentation is performed thoroughly. The results show that the biomass production of WGV10 is always higher than that of W∆SEC5 and W303-1A at all stages of fermentation, and that ethanol production of WGV10 is 1.41-fold higher than that of W303-1A. Obviously, VPS10 overexpression is beneficial for yeast and is a more promising method for reduction of PrA excretion.