Sha Xu

Identification of membrane proteins associated with phenylpropanoid tolerance and transport in Escherichia coli BL21.

UNLABELLED Phenylpropanoids are phytochemicals produced by some plants and possess a wide variety of biological activities. These compounds exist in plants in low amounts. Production of them in genetically engineered microorganisms has many advantages. A majority of functional phenylpropanoids are toxic to microbial hosts. Export of these compounds may relieve the cellular toxicity and increase the yield. However, proteins and mechanisms involved in phenylpropanoids transport and tolerance remain poorly understood. In this study, 16 membrane proteins that were differentially expressed in Escherichia coli in response to three typical phenylpropanoids (resveratrol, naringenin and rutin) were identified using a membrane proteomics approach. These proteins included outer membrane proteins OmpA, OmpF, OmpW, FadL, TolC, LamB, and YaeT, peripheral membrane proteins AtpD, AtpH, YgaU, OppA, MalK, and MalE, and cytoplasmic membrane proteins OppD, PotG, and ManX. Functions of these proteins were determined by using gene overexpression and silencing. The results suggest that OmpA and FadL may play important roles in the transmembrane export of phenylpropanoids in E. coli. LamB, MalE, MalK and ManX may participate in phenylpropanoid uptake. The role of YgaU in enhancing the tolerance to phenylpropanoids remains to be determined. These results may assist the engineering of microorganisms with enhanced phenylpropanoid producing capabilities. BIOLOGICAL SIGNIFICANCE Phenylpropanoids are phytochemicals produced by some plants and possess a wide variety of biological activities. Both the tolerance and the transport of phenylpropanoids play important roles in systematic metabolic engineering of microorganisms to produce these phytochemicals. Both specific and non-specific transporters are essential for these functions but remain poorly understood. This research utilized membrane proteomics to identify E. coli BL21 (DE3) membrane proteins that may be involved in phenylpropanoid transport and tolerance. These results may facilitate the construction of more efficient microbial phenylpropanoid producers through genetic engineering of membrane transporter proteins.

Enhanced production of L-sorbose from D-sorbitol by improving the mRNA abundance of sorbitol dehydrogenase in Gluconobacter oxydans WSH-003

BackgroundProduction of L-sorbose from D-sorbitol by Gluconobacter oxydans is the first step to produce L-ascorbic acid on industrial scale. The sldhAB gene, which encodes the sorbitol dehydrogenase (SLDH), was overexpressed in an industrial strain G. oxydans WSH-003 with a strong promoter, PtufB. To enhance the mRNA abundance, a series of artificial poly(A/T) tails were added to the 3′-terminal of sldhAB gene. Besides, their role in sldhAB overexpression and their subsequent effects on L-sorbose production were investigated.ResultsThe mRNA abundance of the sldhAB gene could be enhanced in G. oxydans by suitable poly(A/T) tails. By self-overexpressing the sldhAB gene in G. oxydans WSH-003 with an optimal poly(A/T) tail under the constitutive promoter PtufB, the titer and the productivity of L-sorbose were enhanced by 36.3% and 25.0%, respectively, in a 1-L fermenter. Immobilization of G. oxydans-sldhAB6 cells further improved the L-sorbose titer by 33.7% after 20 days of semi-continuous fed-batch fermentation.ConclusionsThe artificial poly(A/T) tails could significantly enhance the mRNA abundance of the sldhAB. Immobilized G. oxydans-sldhAB6 cells could further enlarge the positive effect caused by enhanced mRNA abundance of the sldhAB.

Water-forming NADH oxidase protects Torulopsis glabrata against hyperosmotic stress.

A heterologous water‐forming NADH oxidase was introduced into Torulopsis glabrata and the effect on cell growth under hyperosmotic conditions was investigated. Expression of the noxE gene from Lactococcus lactis NZ9000 in T. glabrata resulted in a marked decrease in the NADH : NAD+ ratio and higher activities of key enzymes in water‐regenerating pathways, leading to an increase in intracellular water content. NaCl‐induced reactive oxygen species production was also decreased by the introduction of NADH oxidase, resulting in a significant increase in the growth of T. glabrata under hyperosmotic stress conditions (3824 mOsmol/kg). The results indicated that the osmotolerance of cells can be enhanced by manipulating water‐production pathways. Copyright

Accelerating Glycolytic Flux of Torulopsis glabrata CCTCC M202019 at High Oxidoreduction Potential Created Using Potassium Ferricyanide

This study aimed to increase the glycolytic flux of the multivitamin auxotrophic yeast Torulopsis glabrata by redirecting NADH oxidation from oxidative phosphorylation to membrane‐bound ferric reductase. We added potassium ferricyanide as electron acceptor to T. glabrata culture broth at 20% dissolved oxygen (DO) concentration, which resulted in: (1) decreases in the NADH content, NADH/NAD+ ratio, and ATP level of 45.3%, 60.3%, and 15.2%, respectively; (2) high activities of the key glycolytic enzymes hexokinase, phosphofructokinase, and pyruvate kinase, as well as high expression levels of the genes encoding these enzymes; and (3) increases in the specific glucose consumption rate and pyruvate yield of T. glabrata was by 45.5% and 23.1%, respectively. Our results showed that membrane‐bound ferric reductase offers an alternative and efficient NADH oxidation pathway at lower DO concentration, which increases the glycolytic flux of T. glabrata.

Enhanced pyruvate production in Candida glabrata by carrier engineering

Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria, a high central carbon metabolism rate, specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate‐limiting glycolytic enzymes. Moreover, pyruvate production of CGΔura3‐Sp‐MPC1, CGΔura3‐Sp‐MPC2, and CGΔura3‐Sp‐MPC1‐Sp‐MPC2 increased 134.4%, 120.3%, and 30.0%, respectively. In conclusion, lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms.

Efficient transformation of Rhizopus delemar by electroporation of germinated spores

High efficient transformation of mycelial fungi is essential to both metabolic engineering and physiological analysis of these industrially important microorganisms. However, transformation efficiencies for mycelial fungi are highly restricted by difficulties in colony formation and competent cell preparation. In this work, an innovative transformation procedure that could significantly improve the efficiency of colony formation and transformation process has been established for a typical mycelial fungus, Rhizopus delemar. Single colonies of R. delemar were obtained with the addition of sodium deoxycholate. Fresh germinated spores of R. delemar were successfully transformed by electroporation. In addition, by pretreatment of the germinated spores with 0.05M lithium acetate (LiAc) and 20mM dithiothreitol (DTT) before electroporation, the transformation efficiency was further improved by 9.5-fold. The final transformation efficiency at optimal conditions reached 1239 transformants/μg DNA. The method described here would facilitate more efficient metabolic engineering and investigation of physiological functions in R. delemar or other similar mycelial fungi.

Spatial organization of silybin biosynthesis in milk thistle [Silybum marianum (L.) Gaertn]

Silymarin is a collection of compounds extracted from the medicinal herb milk thistle, among which silybin is the major flavonolignan. However, the biosynthesis pathway of silybin remains unclear. In this study, biomimetic reactions demonstrated that silybin can be synthesized from coniferyl alcohol and taxifolin by the action of peroxidase. The concentration profiles of silybin and its precursors and RNA-Seq analysis of gene expression revealed that the amount of taxifolin and the activity of peroxidase serve as the limiting factors in silybin biosynthesis. Hierarchical clustering of the expression profile of genes of the flavonoid biosynthesis pathway distinguished flowers from other organs. RNA-Seq revealed five candidates for the peroxidase involved in silybin production, among which APX1 (ascorbate peroxidase 1) showed a distinct peroxidase activity and the capacity to synthesize silybin. The spatial organization of silybin biosynthesis in milk thistle was elucidated, which could help our understanding of the biosynthesis of silybin and other flavonolignans.

Complete genome sequence and analysis of the industrial Saccharomyces cerevisiae strain N85 used in Chinese rice wine production

Abstract Chinese rice wine is a popular traditional alcoholic beverage in China, while its brewing processes have rarely been explored. We herein report the first gapless, near-finished genome sequence of the yeast strain Saccharomyces cerevisiae N85 for Chinese rice wine production. Several assembly methods were used to integrate Pacific Bioscience (PacBio) and Illumina sequencing data to achieve high-quality genome sequencing of the strain. The genome encodes more than 6,000 predicted proteins, and 238 long non-coding RNAs, which are validated by RNA-sequencing data. Moreover, our annotation predicts 171 novel genes that are not present in the reference S288c genome. We also identified 65,902 single nucleotide polymorphisms and small indels, many of which are located within genic regions. Dozens of larger copy-number variations and translocations were detected, mainly enriched in the subtelomeres, suggesting these regions may be related to genomic evolution. This study will serve as a milestone in studying of Chinese rice wine and related beverages in China and in other countries. It will help to develop more scientific and modern fermentation processes of Chinese rice wine, and explore metabolism pathways of desired and harmful components in Chinese rice wine to improve its taste and nutritional value.

Separation of α-ketoglutaric acid and pyruvic acid from the culture broth of Yarrowia lipolytica WSH-Z06 by chromatographic methods: Separation of keto acids from the culture broth of Yarrowia lipolytica WSH-Z06.

Both α‐ketoglutaric acid (KGA) and pyruvic acid (PYR) are important keto acids. Efficient co‐production of KGA and PYR has been achieved in our previous work, and could significantly decrease the cost of fermentation production. KGA and PYR have similar physical and chemical properties. Hence, finding a way to separate the two keto acids efficiently has become a key challenge. In this study, different chromatographic methods have been investigated, including ion‐exchange chromatography, aluminum oxide chromatography and silica gel chromatography. The results show that the two keto acids can be well separated with silica gel chromatography, whereas ion‐exchange chromatography and aluminum oxide chromatography could not separate them. Using the pretreated fermentation broth of Yarrowia lipolytica WSH‐Z06, the purity and yield of KGA/PYR reached 98.7%/99.1% and 86.7%/70.9%, respectively, with an optimized silica gel chromatography‐based procedure. This study provides an efficient method for separating KGA and PYR from fermentation broth, which might be applied on an industrial scale and significantly decrease the cost of biotechnological production of keto acids.

Current challenges facing one-step production of l-ascorbic acid

l-ascorbic acid (L-AA, vitamin C) is an essential vitamin that is widely used as a nutrient or medicine in the pharmaceuticals, cosmetics, food, beverage and feed additive industries, and accounts for the largest share of the global vitamins market. L-AA is mainly produced by a classic two-step fermentation process that suffers from the use of a multi-step mixed culture system and two rounds of sterilisation, which significantly increases the cost of the final product. One-step fermentation has been attempted, but a method rivalling the efficiency of the two-step process has not yet been achieved on an industrial scale. In this review, based on the current classical two-step fermentation processes and other potential routes for L-AA production, the challenges and pitfalls of a one-step fermentation process are summarised. The prospects for one-step fermentation production of L-AA and how this might be achieved are also discussed.