The Komagatella phaffii ACG1 gene, encoding ß-1,6-N-acetylglucosaminyltransferase, is involved in the autophagy of cytosolic and peroxisomal proteins.

The methylotrophic yeast Komagataella phaffii is considered one of the most effective producers of recombinant proteins of industrial importance. Effective producers should be characterized by the maximal reduction of degradation of the cytosolic recombinant proteins. The mechanisms of degradation of cytosolic proteins in K. phaffii have not been elucidated; however, data suggest that they are partially degraded in the autophagic pathway. To identify factors that influence this process, a developed system for the selection of recombinant strains of K. phaffii with impaired autophagic degradation of the heterologous model cytosolic protein (yeast ß-galactosidase) was used for insertional tagging of the genes involved in cytosolic proteins degradation. In one of the obtained strains, the insertion cassette disrupted the open reading frame of the gene encoding ß-1,6-N-acetylglucosaminyltransferase. A recombinant strain with deletion of this gene was also obtained. The rate of degradation of the ß-galactosidase enzyme was two times slower in the insertion mutant and 1.5 times slower in the deletion strain as compared to the parental strain with native ß-1,6-N-acetylglucosaminyltransferase. The rate of degradation of native K. phaffii cytosolic and peroxisomal enzymes, formaldehyde dehydrogenase, formate dehydrogenase, and alcohol oxidase, respectively, showed similar trends to that of ß-galactosidase-slower degradation in the deletion and insertional mutants as compared to the wild-type strain, but faster protein degradation relative to the strain completely defective in autophagy. We conclude that K. phaffii gene designated ACG1, encoding ß-1,6-N-acetylglucosaminyltransferase, is involved in autophagy of the cytosolic and peroxisomal proteins.

The role of hexose transporter-like sensor hxs1 and transcription activator involved in carbohydrate sensing azf1 in xylose and glucose fermentation in the thermotolerant yeast Ogataea polymorpha.

BACKGROUND: Fuel ethanol from lignocellulose could be important source of renewable energy. However, to make the process feasible, more efficient microbial fermentation of pentose sugars, mainly xylose, should be achieved. The native xylose-fermenting thermotolerant yeast Ogataea polymorpha is a promising organism for further development. Efficacy of xylose alcoholic fermentation by O. polymorpha was significantly improved by metabolic engineering. Still, genes involved in regulation of xylose fermentation are insufficiently studied. RESULTS: We isolated an insertional mutant of O. polymorpha with impaired ethanol production from xylose. The insertion occurred in the gene HXS1 that encodes hexose transporter-like sensor, a close homolog of Saccharomyces cerevisiae sensors Snf3 and Rgt2. The role of this gene in xylose utilization and fermentation was not previously elucidated. We additionally analyzed O. polymorpha strains with the deletion and overexpression of the corresponding gene. Strains with deletion of the HXS1 gene had slower rate of glucose and xylose consumption and produced 4 times less ethanol than the wild-type strain, whereas overexpression of HXS1 led to 10% increase of ethanol production from glucose and more than 2 times increase of ethanol production from xylose. We also constructed strains of O. polymorpha with overexpression of the gene AZF1 homologous to S. cerevisiae AZF1 gene which encodes transcription activator involved in carbohydrate sensing. Such transformants produced 10% more ethanol in glucose medium and 2.4 times more ethanol in xylose medium. Besides, we deleted the AZF1 gene in O. polymorpha. Ethanol accumulation in xylose and glucose media in such deletion strains dropped 1.5 and 1.8 times respectively. Overexpression of the HXS1 and AZF1 genes was also obtained in the advanced ethanol producer from xylose. The corresponding strains were characterized by 20-40% elevated ethanol accumulation in xylose medium. To understand underlying mechanisms of the observed phenotypes, specific enzymatic activities were evaluated in the isolated recombinant strains. CONCLUSIONS: This paper shows the important role of hexose sensor Hxs1 and transcription factor Azf1 in xylose and glucose alcoholic fermentation in the native xylose-fermenting yeast O. polymorpha and suggests potential importance of the corresponding genes for construction of the advanced ethanol producers from the major sugars of lignocellulose.

100 Years Later, What Is New in Glycerol Bioproduction?

Industrial production of glycerol by yeast, which began during WWI in the so-called Neuberg fermentation, was the first example of metabolic engineering. However, this process, based on bisulfite addition to fermentation liquid, has many drawbacks and was replaced by other methods of glycerol production. Osmotolerant yeasts and other microorganisms that do not require addition of bisulfite to steer cellular metabolism towards glycerol synthesis have been discovered or engineered. Because the glycerol market is expected to reach 5 billion US$ by 2024, microbial fermentation may again become a promising way to produce glycerol. This review summarizes some problems and perspectives on the production of glycerol by natural or engineered eukaryotic and prokaryotic microorganisms.

Overexpression of Transcription Factor ZNF1 of Glycolysis Improves Bioethanol Productivity under High Glucose Concentration and Enhances Acetic Acid Tolerance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae offers an attractive platform for synthesis of biofuels and biochemical; however, robust strains that can withstand high substrate concentration and fermentation conditions are required. To improve the yield and productivity of bioethanol, modification of glucose metabolism and cellular stress adaptation is investigated. Specifically, the role of Znf1 transcription factor in metabolic regulation of glucose is characterized. Here, Znf1 is first shown to activate key genes in glycolysis, pyruvate metabolism, and alcoholic fermentation when glucose is provided as the sole carbon source. Under conditions of high glucose (20 g L-1 ), overexpression of ZNF1 accelerated glucose consumption with only 0.67-0.80% of glucose remaining after 24 or 36 h of fermentation. Importantly, ZNF1 overexpression increases ethanol concentrations by 14-24% and achieves a maximum ethanol concentration of 76.12-88.60 g L-1 . Ethanol productivity is increased 3.17-3.69 in strains overexpressing ZNF1 compared to 2.42-3.35 and 2.94-3.50 for the znf1Δ and wild-type strains, respectively. Moreover, strains overexpressing ZNF1 also display enhanced tolerance to osmotic and weak-acid stresses, important trait in alcoholic fermentation. Overexpresssion of key transcriptional activators of genes in glycolysis and stress responses appears to be an effective strategy to improve bioethanol productivity and enhance strain robustness.

Metabolic engineering for high glycerol production by the anaerobic cultures of Saccharomyces cerevisiae.

Glycerol is used by the cosmetic, paint, automotive, food, and pharmaceutical industries and for production of explosives. Currently, glycerol is available in commercial quantities as a by-product from biodiesel production, but the purity and the cost of its purification are prohibitive. The industrial production of glycerol by glucose aerobic fermentation using osmotolerant strains of the yeasts Candida sp. and Saccharomyces cerevisiae has been described. A major drawback of the aerobic process is the high cost of production. For this reason, the development of yeast strains that effectively convert glucose to glycerol anaerobically is of great importance. Due to its ability to grow under anaerobic conditions, the yeast S. cerevisiae is an ideal system for the development of this new biotechnological platform. To increase glycerol production and accumulation from glucose, we lowered the expression of TPI1 gene coding for triose phosphate isomerase; overexpressed the fused gene consisting the GPD1 and GPP2 parts coding for glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, respectively; overexpressed the engineered FPS1 gene that codes for aquaglyceroporin; and overexpressed the truncated gene ILV2 that codes for acetolactate synthase. The best constructed strain produced more than 20 g of glycerol/L from glucose under micro-aerobic conditions and 16 g of glycerol/L under anaerobic conditions. The increase in glycerol production led to a drop in ethanol and biomass accumulation.

Overexpression of the genes PDC1 and ADH1 activates glycerol conversion to ethanol in the thermotolerant yeast Ogataea (Hansenula) polymorpha.

Conversion of byproduct from biodiesel production glycerol to high-value compounds is of great importance. Ethanol is considered a promising product of glycerol bioconversion. The methylotrophic thermotolerant yeast Ogataea (Hansenula) polymorpha is of great interest for this purpose as the glycerol byproduct contains methanol and heavy metals as contaminants, and this yeast utilizes methanol and is relatively resistant to heavy metals. Besides, O. polymorpha shows robust growth on glycerol and produces ethanol from various carbon sources. The thermotolerance of this yeast is an additional advantage, allowing increased fermentation temperature to 45-48 °C, leading to increased rate of the fermentation process and a fall in the cost of distillation. The wild-type strain of O. polymorpha produces insignificant amounts of ethanol from glycerol (0.8 g/l). Overexpression of PDC1 coding for pyruvate decarboxylase enhanced ethanol production up to 3.1 g/l, whereas simultaneous overexpression of PDC1 and ADH1 (coding for alcohol dehydrogenase) led to further increase in ethanol production from glycerol. Moreover, the increased temperature of fermentation up to 45 °C stimulated the production of ethanol from glycerol used as the only carbon source up to 5.0 g/l, which exceeds the data obtained by methylotrophic yeast strains reported so far. Copyright © 2016 John Wiley & Sons, Ltd.

Activation of futile cycles as an approach to increase ethanol yield during glucose fermentation in Saccharomyces cerevisiae.

An increase in ethanol yield by yeast from the fermentation of conventional sugars such as glucose and sucrose is possible by reducing the production of a key byproduct such as cellular biomass. Previously we have reported that overexpression of PHO8 gene encoding non-specific ATP-hydrolyzing alkaline phosphatase can lead to a decrease in cellular ATP content and to an increase in ethanol yield during glucose fermentation by Saccharomyces cerevisiae. In this work we further report on 2 new successful approaches to reduce cellular levels of ATP that increase ethanol yield and productivity. The first approach is based on the overexpression of the heterologous Escherichia coli apy gene encoding apyrase or SSB1 part of the chaperon that exhibit ATPase activity in yeast. In the second approach we constructed a futile cycle by the overexpression of S. cerevisiae genes encoding pyruvate carboxylase and phosphoenolpyruvate carboxykinase in S. cerevisiae. These genetically engineered strains accumulated more ethanol compared to the wild-type strain during alcoholic fermentation.

Development of a system for multicopy gene integration in Saccharomyces cerevisiae.

In this study we describe construction and evaluation of a vector for multicopy integration in yeast Saccharomyces cerevisiae. In this vector a modified selective marker and a reporter gene PHO8 (encoding alkaline phosphatase) were flanked with delta sequences of the Ty1 transposon. Modified by error-prone PCR version of selection marker kanMX4 was obtained from Escherichia coli clone with impaired geneticin (G418) resistance. The attenuation of kanMX4 gene provides an opportunity to select for explicitly multicopy integration of the module in S. cerevisiae using moderate (200 mg L(-1)) antibiotic concentrations. The developed system provided integration of 3-10 copies of the module in the genome of S. cerevisiae. High copy integration events were confirmed by qRT-PCR, Southern hybridization and reporter enzyme activity measurements.

Zinc cluster protein Znf1, a novel transcription factor of non-fermentative metabolism in Saccharomyces cerevisiae.

The ability to rapidly respond to nutrient changes is a fundamental requirement for cell survival. Here, we show that the zinc cluster regulator Znf1 responds to altered nutrient signals following glucose starvation through the direct control of genes involved in non-fermentative metabolism, including those belonged to the central pathways of gluconeogenesis (PCK1, FBP1 and MDH2), glyoxylate shunt (MLS1 and ICL1) and the tricarboxylic acid cycle (ACO1), which is demonstrated by Znf1-binding enrichment at these promoters during the glucose-ethanol shift. Additionally, reduced Pck1 and Fbp1 enzymatic activities correlate well with the data obtained from gene transcription analysis. Cells deleted for ZNF1 also display defective mitochondrial morphology with unclear structures of the inner membrane cristae when grown in ethanol, in agreement with the substantial reduction in the ATP content, suggesting for roles of Znf1 in maintaining mitochondrial morphology and function. Furthermore, Znf1 also plays a role in tolerance to pH and osmotic stress, especially during the oxidative metabolism. Taken together, our results clearly suggest that Znf1 is a critical transcriptional regulator for stress adaptation during non-fermentative growth with some partial overlapping targets with previously reported regulators in Saccharomyces cerevisiae.

Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase.

BACKGROUND: The production of ethyl alcohol by fermentation represents the largest scale application of Saccharomyces cerevisiae in industrial biotechnology. Increased worldwide demand for fuel bioethanol is anticipated over the next decade and will exceed 200 billion liters from further expansions. Our working hypothesis was that the drop in ATP level in S. cerevisiae cells during alcoholic fermentation should lead to an increase in ethanol production (yield and productivity) with a greater amount of the utilized glucose converted to ethanol. Our approach to achieve this goal is to decrease the intracellular ATP level via increasing the unspecific alkaline phosphatase activity. RESULTS: Intact and truncated versions of the S. cerevisiae PHO8 gene coding for vacuolar or cytosolic forms of alkaline phosphatase were fused with the alcohol dehydrogenase gene (ADH1) promoter. The constructed expression cassettes used for transformation vectors also contained the dominant selective marker kanMX4 and S. cerevisiae δ-sequence to facilitate multicopy integration to the genome. Laboratory and industrial ethanol producing strains BY4742 and AS400 overexpressing vacuolar form of alkaline phosphatase were characterized by a slightly lowered intracellular ATP level and biomass accumulation and by an increase in ethanol productivity (13% and 7%) when compared to the parental strains. The strains expressing truncated cytosolic form of alkaline phosphatase showed a prolonged lag-phase, reduced biomass accumulation and a strong defect in ethanol production. CONCLUSION: Overexpression of vacuolar alkaline phosphatase leads to an increased ethanol yield in S. cerevisiae.