Regulation of thiamine synthesis in Saccharomyces cerevisiae for improved pyruvate production

Metabolic engineering of Saccharomyces cerevisiae for high‐yield production of carboxylic acid requires a cytosolic pyruvate pool as precursor. In this study, a novel strategy to improve pyruvate production and reduce metabolic by‐products via regulating thiamine synthesis was explored. Two of the thiamine biosynthesis regulatory genes, THI2 and THI3, were disrupted in the S. cerevisiae parent strain FMME‐002. The mutants FMME‐002ΔTHI2 and FMME‐002ΔTHI3 both exhibited an enhanced pyruvate yield. Moreover, FMME‐002ΔTHI2 achieved a relatively higher pyruvate production, and the highest concentration of pyruvate was achieved when 0.04 µ m thiamine was added. Enzyme assays and fermentation profiles of the THI2‐complemented strain indicated that the observed metabolic changes represented intrinsic effects of THI2 deletion on the physiology of S. cerevisiae. Under optimal C:N ratio conditions, FMME‐002ΔTHI2 produced pyruvate up to 8.21 ± 0.30 g/l, whereas the ethanol titre decreased to 2.21 ± 0.24 g/l after 96 h of cultivation. These results demonstrate the possibility of improving pyruvate production by regulating thiamine synthesis in S. cerevisiae. Copyright © 2012 John Wiley & Sons, Ltd.     

Regulation of THI4(MOL1), a thiamine-biosynthetic gene of Saccharomyces cerevisiae

THI4, a Saccharomyces cerevisiae gene originally identified as a result of transient expression in molasses medium and named MOL1 is regulated by thiamine. Using a THI4 promoter-lacZ fusion on a centromeric yeast vector, we have shown that the THI4 is completely repressed throughout batch culture by thiamine at a concentration around 1 μM, but shows high level constitutive expression in thiamine-free medium. The transient expression pattern observed in molasses medium can be mimicked by the addition of 0·15 μM-thiamine to defined minimal medium. Cells grown in thiamine-free medium have an intracellular thiamine concentration of around 9 pmol/10⁷ cells. A low level (1 μM) of exogenous thiamine is completely sequestered from the medium within 30 min; intracellular thiamine concentrations rise rapidly, followed by a gradual decrease as a result of dilution during growth. A saturating extracellular level of thiamine leads to a maximal intracellular concentration of around 1600 pmol/10⁷ cells, at which point the transport system is shut down. After transfer from repressing to non-repressing medium, THI4 becomes induced when the intracellular concentration of thiamine falls to 20 pmol/10⁷ cells. A thi4::UARA3 disruption strain is auxotrophic for thiamine, but can grow in the presence of hydroxyethyl thiazole, indicating that the gene product is involved in the biosynthetic pathway leading to the formation of the thiazole precursor of thiamine.     

Isolation of glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae and their use in baker's yeast production

 Glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae 0511 were obtained by mutation under specific conditions. A total of 3388 strains were isolated and among them were found 46 mutants sensitive to methylglyoxal. The intracellular glutathione concentration of mutant strain S. cerevisiae 3033 was 0.0172 g/g dry weight, which was a decrease of >76% compared to that of the parent. The growth of mutant strains S. cerevisiae 3033 and S. cerevisiae 1116 in the medium with glutathione present and absent was compared to that of the parent strain. The sensibility of the baker's yeast strains studied to antifoaming agents, butanol and acetic acid was also investigated. The relationship between glutathione presence in the cell and the sensitivity of strain S. cerevisiae 3033 to antifoaming agents and butanol was ascertained, while such a connection with the presence of acetic acid in the molasses medium used for baker's yeast cultivation was not observed. The higher sensitivity of strain S. cerevisiae 3033 to some chemical compounds in the molasses nutrition medium was shown. Received: 2 November 1999 / Revised version: 15 February 2000     

YEAST MUTANTS EXCRETING VITAMIN B1 AND THEIR USE IN THE PRODUCTION OF THIAMINE RICH BEERS*,†

In certain strains of Saccharomyces cerevisiae and Saccharomyces uvarum (carlsbergensis), high doses of ultraviolet radiation induced stable mutants excreting thiamine from their living cells during the ethanol production. In S. cerevisiae, the first mutagenesis step yielded mutants with the production of 0·3–0·5 mg extracellular thiamine. HCL/litre minimum medium without thiamine and the second one increased the production up to 2·0–2·1 mg thiamine. HCL/litre. In S. uvarum (carlsbergensis), mutants producing up to 0·76 mg thiamine. HCL/litre 10% Plato hopped wort were obtained in the first mutagenesis step combined with mitotic recombinations. The increase up to 0·92 mg/litre was achieved here by repeated selections. Both laboratory and pilot plant fermentations in 10 and 4% Plato hopped worts showed the suitability of selected mutants for the production of thiamine rich beers which fulfilled all quality requirements and contained 0·67–0·80 and 0·22–0·33 mg thiamine. HCI/litre, respectively.     

VITAMIN REQUIREMENTS OF CERTAIN PEDIOCOCCI ISOLATED FROM BREWERY PRODUCTS

A medium has been developed for determination of the vitamin requirements of a number of pedicocci, most of which were isolated from brewery products and classified as Pediococcus damnosus. Tween 80 was essential to a number of the P. damnosus strains in the medium used. Folic acid was active only on a test strain, P. cerevisiae NCTC 8066. Biotin had a strong growth-promoting effect on most of the strains. Pantothenic acid was essential or highly stimulating to the growth of all strains. Riboflavin is considered essential to the P. damnosus strains, but was inactive with the two test strains used, P. cerevisiae NCTC 8066 and P. acidilactici NCIB 6990. Pyridoxin had a growth-promoting effect on most of the strains, and was essential to one P. damnosus strain. Ascorbic acid was inactive on the P. cerevisiae and P. acidilactici strains, had a slight growth-promoting effect on one P. damnosus strain, but inhibited, partly or completely, the growth of the other strains. Ascorbic acid was therefore omitted from the vitamin medium. p-Aminobenzoic acid, vitamin B-12 (cyanocobalamin), thiamine, nicotinic acid, nicotinamide, retinol acetate and c. 0·5% v/v alcohol were inactive on all the pediococcus strains studied.     

Mapping of gene controlling thiamine transport in Saccharomyces cerevisiae

A recessive mutation leading to complete loss of thiamine uptake in Saccharomyces cerevisiae was mapped on the left arm of chromosome VII, approximately 56cM centromere-distal to trp5. As the analysed locus is relatively distant from its centromere and from the markers used, its attachment to chromosome VII was confirmed by chromosome loss methods.     

IV. Yeast sequencing reports. Nucleotide sequence of the SAC2 gene of Saccharomyces cerevisiae

A temperature-sensitive mutation (act1-1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC2 gene. A cloned genomic DNA fragment that complements the cold-sensitive growth phenotype associated with such a suppressor mutation (sac2-1) was sequenced. The fragment contained an open reading frame that encodes a 641 amino acid predicted hydrophilic protein with a molecular weight of 74 445. No sequences with significant similarity to SAC2 were found in the GenBank and EMBL databases. A SAC2 disruption mutation was constructed which had phenotypes similar to the sac2-1 point mutation. A haploid SAC2 disruption strain failed to grow at low temperature and the disruption allele suppressed the temperature-sensitive act1-1 growth defect. The suppression phenotype was dependent on the strain background. The SAC2 sequence has been submitted to the EMBL data library (Accession Number Z29988).     

GUT2, a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae

A gut2 mutant of Saccharomyces cerevisiae is deficient in the mitochondrial glycerol 3-phosphate dehydrogenase and hence cannot utilize glycerol. Upon transformation of a gut2 mutant strain with a low-copy yeast genomic library, hybrid plasmids were isolated which complemented the gut2 mutation. The nucleotide sequence of a 3·2 kb PstI-XhoI fragment complementing a gut2 mutant strain is presented. The fragment reveals an open reading frame (ORF) encoding a polypeptide with a predicted molecular weight of 68·8 kDa. Disruption of the ORF leads to a glycerol non-utilizing phenotype. A putative flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins. The cloned gene has been mapped both physically and genetically to the left arm of chromosome IX, where the original gut2 mutation also maps. We conclude that the presented ORF is the GUT2 gene and propose that it is the structural gene for the mitochondrial glycerol 3-phosphate dehydrogenase.     

类金属硫蛋白产生菌的诱变育种

本研究选取酿酒酵母Cu-3作为产类金属硫蛋白的出发菌株，采用物理（UV诱变）以及化学诱变（NTG诱变）方法进行复合交替诱变，再进行类金属硫蛋白诱导实验，得到突变菌株N-1，其类金属硫蛋白Cu—MT产量有了十分明显的提高，并且具有很好的遗传稳定性。     

Low-Affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae,a homologue of the Kluyveromyces lactis RAG1 gene

A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.     

产纤维素酶细菌的选育

以产纤维素酶芽孢杆菌JJQ8为出发菌株，进行了紫外线和HNO2诱变处理，采用透明圈法初筛和摇瓶培养复筛，获得2株高产纤维素酶的突变株HS—T001和HS-T002。与出发菌株相比，经紫外线诱变处理的HS．T001突变株产酶活力提高了1．9倍，用HNO2诱变处理的HS—T002突变株产酶活力提高了2倍。     

STUDY OF THE PHENOMENON OF AGGLOMERATION IN THE YEAST SACCHAROMYCES CEREVISIAE

Agglomeration or “grittiness” is detrimental to bakers' yeast quality. Gritty yeast only partially resuspends when mixed in water, most of it remaining as macroscopic cell aggregates. A macroscopic sedimentation test was developed for measuring agglomeration intensity. Expression of the gritty phenotype was investigated in two strains (N176 and GB1) of Saccharomyces cerevisiae grown on a 14-liter scale by varying fermentation conditions of agitation and aeration. Results show that yeast agglomeration is different from yeast flocculation, and is determined by both strain genetic background and environmental factors. The gritty phenotype was expressed in the strain prone to agglomeration (N176) when dissolved oxygen was limiting in the fermenter. Gritty cells had a lower phosphorus and lipid concentration and a higher protein concentration at the surface of the cell, and a higher amount of whole cell and cell wall proteins and calcium than non-gritty cells. Some proteins were also extracted from gritty cells with sodium hydroxide or mercaptoethanol, that were not present in non-gritty cells. Agglomeration did not result in major differences in the structure or composition of the structural cell wall mannoprotein (CWMP). A model for agglomeration is proposed: proteins (cognors) activated by Ca²⁺ (cofactors) to increase their binding capacity bind the mannans (cognons) of adjoining cells; binding is facilitated by the lower phosphorus and lipid concentration at the surface of gritty cells.     

Diversity of urinary excretion patterns of main ellagitannins' colonic metabolites after ingestion of tropical highland blackberry (Rubus adenotrichus) juice

Tropical highland blackberries are a rich source of ellagitannins (ETs), which are metabolized by gut microbiota to yield urolithin, a potentially bioactive compound excreted in urine up to 7 days after ingestion. Following the ingestion of 250 mL of tropical highland blackberry juice, a spot of urine from 26 volunteers collected at 51 ± 4 h was analyzed for urolithin A and B main derivatives (aglycones and glucuronated forms). Three main groups, “no or low urolithin excreters,” “predominantly UA derivatives excreters” and “predominantly UB derivatives excreters,” were observed. These categories were also unambiguously observed from 9 individuals following the total excretion of ETs' main metabolites over a 4-day period after ingesting one shot of blackberry juice. Although relatively high inter- and intra-individual variabilities were observed, individuals preserved their status during different intervention periods with different amounts of ETs ingested. Accurate UPLC-DAD/ESI-Q-TOF/MS2 allowed the tentative assignment of an identity to 15 other ET metabolites in urine, but profiling did not allow the discrimination of any other compounds aside from UA or UB derivatives. The results highlight the importance of the interaction of gut microbiota composition and the host endogenous excretery system, which may play a major role in the observed inter-individual variability.     

Use of PCR‐DHPLC (Polymerase Chain Reaction—Denaturing High Performance Liquid Chromatography) for the Rapid Differentiation of Industrial Saccharomyces pastorianus and Saccharomyces cerevisiae Strains

Strain specific detection and control of Saccharomyces pastorianus and Saccharomyces cerevisiae starter cultures is of great importance for the fermentation industry. The preconditions of strain specific fermentation characteristics can be ensured by periodic analysis and confirmation of the strain identity. With regard to industrial S. pastorianus and S. cerevisiae strains and a focus on brewing strains, the differentiation methods most available are time‐consuming and not very discriminative. In this work PCR‐DHPLC analysis was investigated as a novel approach for the differentiation of industrially used S. pastorianus and S. cerevisiae strains. The PCR‐DHPLC‐system was specific for S. cerevisiae strains and S. pastorianus hybrid strains that contain IGS2 rDNA, which originates from the S. cerevisiae ancestor. For the DNA of 177 strains of 41 non‐target species, which are typical for beverage and fermentation surroundings, the absence of PCR‐amplificates could be confirmed by DHPLC analysis. It was shown that single strains of S. cerevisiae and S. pastorianus could be differentiated. A strain specific differentiation within the group of top‐fermenting Saccharomyces cerevisiae strains could also be performed. For the group of bottom fermenting S. pastorianus brewing strains, strain‐to‐strain specific differences in the DHPLC chromatograms could be observed which can be used to differentiate and to compare two single strains with each other, although the comparison of chromatograms of an unknown S. pastorianus strain with a set of known S. pastorianus chromatograms could only reveal tendencies towards grouping into types. The differential DHPLC chromatogram characteristics (fluorescence intensities, number of peaks/side‐peaks/peak‐shoulders) within S. pastorianus are present, but not as distinctive as for S. cerevisiae. Additionally PCR‐DHPLC has advantages compared to other differentiation methods, such as species specificity, speed (2.5 h for one sample) and precision with the described limits.     

Relationship between growth of food‐spoilage yeast in high‐sugar environments and sensitivity to high‐intensity pulsed UV light irradiation

The relationship between prior growth of food‐spoilage yeast in high‐sugar environments and their subsequent survival postpulsed UV (PUV) irradiation was investigated. Test yeast were separately grown to early stationary phase in YPD broth containing increasing concentrations of glucose (1–50% w/v) and were flashed with ≤40 pulses of broad‐spectrum light at lamp discharge energy settings of 3.2, 7.2 and 12.8 J (equivalent to UV doses of 0.53, 1.09 and 3.36 μJ cm^?2, respectively) and their inactivation measured. Findings showed that prior growth in high‐sugar conditions (≥30% glucose w/v) enhanced the sensitivity of all nine representative strains of Zygosaccharomyces bailii, Z. rouxii and Saccharomyces cerevisiae yeast to PUV irradiation. Significant differences in inactivation amongst different yeast types also occurred depending on amount of UV dose applied, where the order of increasing sensitivity of osmotically stressed yeast to PUV irradiation was shown to be Z. rouxii, Z. bailii and >S. cerevisiae. For example, a 1.2‐log order difference in CFU mL^?1 reduction occurred between Z. bailii 11 486 and S. cerevisiae 834 when grown in 50% w/v sugar samples and treated with the uppermost test UV dosage of 3.36 μJ cm^?2, where these two yeast strains were reduced by 3.8 and 5.0 log orders, respectively, after this PUV treatment regime compared to untreated controls. The higher the UV dose applied the greater the reduction in yeast numbers. For example, a 1.0‐, 1.4‐ and 4.0‐log order differences in CFU mL^?1 numbers occurred for S. cerevisiae 834 grown in 15% w/v sugar samples and then treated with PUV dose of 0.53, 1.09 and 3.36 μJ cm^?2, respectively. These findings support the development of PUV for the treatment of high‐sugar foods that are prone to spoilage by osmotolerant yeast.     

Effect of citrulline metabolism in Saccharomyces cerevisiae on the formation of ethyl carbamate during Chinese rice wine fermentation

Urea, as the main precursor of ethyl carbamate (EC), has received extensive attention. Here, we have metabolically engineered an industrial yeast strain – Saccharomyces cerevisiae N85 – to investigate the contribution of the EC precursor citrulline to the concentration of EC in Chinese rice wine. The results showed that the citrulline biosynthetic pathway of the modified strain N85‐arg3 was completely suppressed by deletion of ARG3, encoding ornithine carbamoyltransferase. However, there were no significant differences in the levels of citrulline or EC between N85‐arg3 and the parental strain N85 during fermentation. In addition, we over‐expressed ARG1 (encoding argininosuccinate synthase) and ARG4 (encoding argininosuccinate lyase) to construct the engineered strains N85^ARG1,4 and N85^ARG1,4‐arg3. The citrulline contents in Chinese rice wine fermented with N85^ARG1,4 and N85^ARG1,4‐arg3 were respectively 24.1 and 20.4% less than that of N85. However, the contents of EC were 23.8 and 28.5% more than that of N85. These results suggested that reducing the formation of EC during Chinese rice wine fermentation by genetically engineering citrulline metabolism in S. cerevisiae was not a viable proposition. Copyright © 2018 The Institute of Brewing & Distilling     

REDUCTION OF SACCHAROMYCES CEREVISIAE, ESCHERICHIA COLI AND LISTERIA INNOCUA IN APPLE JUICE BY ULTRAVIOLET LIGHT

Apple juice was inoculated separately with Saccharomyces cerevisiae, Listeria innocua (ATCC 51742) or Escherichia coli (ATCC 11775) for treatment in a double tube ultraviolet (UV) disinfection system. The apple juice was treated at six flow rates (0.073–0.548 L/min) for selected fluences (75–450 kJ/m²). The juice was also inoculated with a mixture of these three microorganisms and UV light treated from 0.548 to 0.735 L/min for 30 min. The microbial reduction was described with a first order kinetic model. Average D_uvvalues of 23.1–40.5, 8.2–20.6 and 6.0–17.7 min were obtained for S. cerevisiae, L. innocua and E. coli, respectively. A linear model was used to describe the relationship between log D_uvversus flow rate for S. cerevisiae and L. innocua. However, a third order polynomial model was more adequate for describing the E. coli D_uvvalues versus flow rate. Less than 10 (no growth), 190 and 200 cfu/mL of S. cerevisiae, L. inocua and E. coli, respectively, were observed in UV‐treated apple juice inoculated with a mixture of microorganisms.     

EXPRESSION OF THE STA2 GLUCOAMYLASE GENE OF SACCHAROMYCES CEREVISIAE IN BREWERS' YEAST

Two fragments of DNA containing the Saccharomyces cerevisiae STA2 glucoamylase gene, with differing lengths of 5î non-coding DNA, were separately subcloned into a yeast centromeric plasmid. Of these two subclones, only the shorter one (containing 127 base-pairs of 5î non-coding DNA) was able to confer glucoamylase production on a standard laboratory strain of S. cerevisiae. The longer subclone (containing 465 bp of 5î non-coding DNA) did, however, confer glucoamylase production on a strain of S. cerevisiae lacking a functional STA10 gene (which encodes a repressor of STA2 gene expression). All-yeast plasmids lacking bacterial DNA were constructed from the two STA2 subclones for the transformation of a lager brewing yeast. Only the shorter STA2 subclone conferred glucoamylase activity on this yeast. The level of enzyme activity was comparable to that produced by the same yeast strain containing STA2 expressed from the PGK1 (that is, PGK1) promoter.     

THE GENETIC MANIPULATION OF KILLER CHARACTER INTO BREWING YEAST

A procedure is described whereby the cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae is transferred to a brewing yeast strain. Neither preparation of protoplasts of the brewing yeast nor mutation of its nuclear genes are required for this process. The brewing yeast killer strains produced have the advantages over their parent brewing cell that they kill sensitive yeasts and are immune to the killing action of certain killer yeasts. The method described offers significant advantages over the process of transformation as a means of genetically manipulating commercial yeasts.