Methylotrophic yeasts are considered as promising cell factories for bio-manufacturing due to their several advantages such as tolerance to low pH and high temperature. In particular, their methanol utilization ability may help to establish a methanol biotransformation process, which will expand the substrate resource for bio-refinery and the product portfolio from methanol. This review summarize current progress on engineering methylotrophic yeasts for production of proteins and chemicals, and compare the strengths and weaknesses with the model yeast Saccharomyces cerevisiae. The challenges and possible solutions in metabolic engineering of methylotrophic yeasts are also discussed. With the developing efficient genetic tools and systems biology, the methylotrophic yeasts should play more important roles in future green bio-manufacturing.
Metabolic Engineering ; Methanol ; Saccharomyces cerevisiae/genetics* ; Yeasts

Non-conventional yeasts such as Yarrowia lipolytica, Pichia pastoris, Kluyveromyces marxianus, Rhodosporidium toruloides and Hansenula polymorpha have proven to be efficient cell factories in producing a variety of natural products due to their wide substrate utilization spectrum, strong tolerance to environmental stresses and other merits. With the development of synthetic biology and gene editing technology, metabolic engineering tools and strategies for non-conventional yeasts are expanding. This review introduces the physiological characteristics, tool development and current application of several representative non-conventional yeasts, and summarizes the metabolic engineering strategies commonly used in the improvement of natural products biosynthesis. We also discuss the strengths and weaknesses of non-conventional yeasts as natural products cell factories at current stage, and prospects future research and development trends.
Yeasts/genetics* ; Yarrowia/metabolism* ; Gene Editing ; Metabolic Engineering

OBJECTIVETo identify the yeast strains isolated from Massa Medicata Fermentata samples that sold in markets.

METHODThe strains were identified through conventional classification methods including colony characteristics, cell morphology, physiological and biochemical properties, as well as 26S rDNA sequence analysis.

RESULTThe isolated strains Y1, Y3, Y4, Y5 were Cryptococcus albidus, Saccharomyces cerevisiae, Pichia kudriavzevii, Endomyces fibuliger, respectively.

CONCLUSIONAfter fermentation the Massa Medicata Fermentata samples contained a variety of yeast species. Yeasts were the main contribution microorganism of the fermentation process.

Fermentation ; Medicine, Chinese Traditional ; Yeasts ; chemistry ; classification ; genetics ; isolation & purification

Yeast surface display involves that the exogenous protein, which was fused with the yeast outer shell cell wall protein, was genetically anchored on the yeast cell surface. It has been widely used in expression and screening of functional protein. Here, we focused on the construction of lipase-displaying systems and its application in enzymatic biosynthesis, such as fatty acid methyl esters, short-chain flavour esters and sugar esters applications, and so on.
Candida ; enzymology ; genetics ; Lipase ; biosynthesis ; genetics ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Solvents ; Yeasts ; enzymology ; genetics

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.
Gene Editing ; Metabolic Engineering ; Pichia/genetics* ; Synthetic Biology ; Yarrowia/genetics* ; Yeasts

Cloning, Molecular ; DNA ; genetics ; DNA-Binding Proteins ; genetics ; Humans ; Proteins ; genetics ; Sequence Analysis, DNA ; Two-Hybrid System Techniques ; Yeasts ; genetics

OBJECTIVETo screen and clone the genes of proteins in hepatocytes interacting with hepatitis B virus (HBV) PreS2 by yeast-two hybridization technique.

METHODSThe HBV PreS2 gene was amplified by polymerase chain reaction (PCR) and HBV PreS2 bait plasmid was constructed by using yeast-two hybridization system 3, then transformed into yeast AH109, followed by mating with yeast Y187 containing liver cDNA library plasmid in 2 YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) and synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) containing X-alpha-gal for selecting positive blue clones, then amplified by PCR, sequenced, and performed bioinformatics analysis.

RESULTSHBV PreS2 gene was cloned successfully and expressed in yeast AH109.Twenty-six positive colonies were selected, among them, twelve containing metallothionein 2A, one cytochrome C oxidase II, two cytochrome P450 subfamily IV4F, two cytochrome c oxidase subunit 4 isoform 1, three albumin (ALB), one Na(+)K(+) transporting ATPase beta-1 polypeptide, two prealbumin, one lectin galactoside-binding subunit, and Two new genes with unknown function.

CONCLUSIONGenes of HBV PreS2 interacting proteins have been successfully cloned, which brings some new clues for studying the biological functions of HBV PreS2 and related proteins.

Cloning, Molecular ; Hepatitis B Surface Antigens ; genetics ; physiology ; Plasmids ; Protein Precursors ; genetics ; physiology ; Two-Hybrid System Techniques ; Yeasts ; genetics

Cloning, Molecular ; Gene Expression ; Liver ; physiology ; Liver Regeneration ; Recombinant Proteins ; genetics ; metabolism ; Yeasts

Antimicrobial peptides (AMPs), a class of short proteins with a broad spectrum of antibacterial activities, are isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. They are a key component of the innate immune response in most multicellular organisms. Owing to their potent, broad-spectrum antibacterial activities and uneasy developing of drug resistance, these peptides are of great clinical significance. However, preparation of AMPs at a large scale is a severe challenge to the development of the commercial products. Undoubtedly, construction of high-level biological expression systems for the production of AMPs is the key in its clinical application process. Herein, we summarize the progress in researches on heterogenous expression of AMPs in prokaryotic expression systems and eukaryotic expression systems.
Animals ; Antimicrobial Cationic Peptides ; biosynthesis ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Insecta ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Yeasts ; genetics ; metabolism

Glutamine is an important conditionally necessary amino acid in human body. The effort is to establish a new and high efficient L-glutamine production system instead of traditional fermentaion. In this paper, high efficiency of L-glutamine production is obtained by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system. Glutamine Synthetase gene (glnA) was amplified from Bacillus subtilis genomic DNA with primers designed according to sequences reported in EMBL data bank, then it was inserted into expression vector PET28b, the sequence of glnA was proved to be the same as that reported in the data bank by DNA sequencing. After transformation of this recombinant plasmid PET28b-glnA into BL-21 (DE3) strain, Lactose and IPTG were used to induce GS expression at 37 degrees C separately. Both of them can induce GS expression efficiently. The induced protein is proved to be soluble and occupies about 80% of the total proteins by SDS-PAGE analysis. The soluble GS was purified by Ni2+ chelating sepharose colum. After purification, the purified enzyme was proved active. Results reveal that the optmum temperature of this enzyme is 60 degrees C and optmum pH is 6.5 in biosynthetic reaction by using glutamate, ammonium choloride and ATP as substrates. After induction, the enzyme activity in crude extract of BL-21/PET28b-glnA is 83 times higher than that of original BL-21 extract. Mn2+ can obviously increase the activity and stability of this enzyme. Experiments show that the transformation efficiency of glutamate to glutamine is more than 95%. Because of the high cost from ATP, a system coupling GS with yeast for ATP regenaration was established. In this system, GS utilizes ATP released by yeast fermentation to synthesize L-glutamine. Yeast was treated by 2% toluence to increase its permeability and a yeast named YC001 with high yield of glutamine by coupling with recombinant GS was obtained. The good efficiency was achieved with the presence of 250 mmol/L glucose and 200 mmol/L phosphate, the transformation efficiency of glutamate to glutamine in this system is more than 80%, the average yield of glutamine is about 22g/L. This provides the basis for future large scale production of L-glutamine.
Bacillus subtilis ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; methods ; Glutamate-Ammonia Ligase ; biosynthesis ; genetics ; Glutamic Acid ; metabolism ; Glutamine ; biosynthesis ; genetics ; Yeasts ; genetics ; metabolism