Large Flux Supply of NAD(H) under Aerobic Conditions by Candida glycerinogenes.

NAD is a redox coenzyme and is the center of energy metabolism. In metabolic engineering modifications, an insufficient NAD(H) supply often limits the accumulation of target products. In this study, Candida glycerinogenes was found to be able to supply NAD(H) in large fluxes, up to 7.6 times more than Saccharomyces cerevisiae in aerobic fermentation. Aerobic fermentation in a medium without amino nitrogen sources demonstrated that C. glycerinogenes NAD synthesis was not dependent on NAD precursors in the medium. Inhibition by antisense RNA and the detection of transcript levels indicated that the main NAD supply pathway is the de novo biosynthesis pathway. It was further demonstrated that NAD(H) supply was unaffected by changes in metabolic flow through C. glycerinogenes ΔGPD aerobic fermentation (80 g/L ethanol). In conclusion, the ability of C. glycerinogenes to supply NAD(H) in large fluxes provides a new approach to solving the NAD(H) supply problem in synthetic biology.

Glycerol Production from Undetoxified Lignocellulose Hydrolysate by a Multiresistant Engineered Candida glycerinogenes.

Glycerol is an important platform compound with multidisciplinary applications, and glycerol production using low-cost sugar cane bagasse hydrolysate is promising. Candida glycerinogenes, an industrial yeast strain known for its high glycerol production capability, has been found to thrive in bagasse hydrolysate obtained through a simple treatment without detoxification. The engineered C. glycerinogenes exhibited significant resistance to furfural, acetic acid, and 3,4-dimethylbenzaldehyde within undetoxified hydrolysates. To further enhance glycerol production, genetic modifications were made to Candida glycerinogenes to enhance the utilization of xylose. Fermentation of undetoxified bagasse hydrolysate by CgS45 resulted in a glycerol titer of 40.3 g/L and a yield of 40.4%. This process required only 1 kg of bagasse to produce 93.5 g of glycerol. This is the first report of glycerol production using lignocellulose, which presents a new way for environmentally friendly industrial production of glycerol.

Verification of a novel glyceraldehyde-3-phosphate dehydrogenase capable of histamine degradation and its preliminary application in wine production.

The search for enzymes with histamine-degrading activity is of great interest, since it has great potential in the way of solving the problem of high histamine levels in food. In this study, the gene of a novel histamine-degrading enzyme, i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Lactobacillus plantarum was cloned and successfully expressed in Escherichia coli DH5α, with the recombinant host determined as histamine-degrading active. The recombinant GAPDH was then purified to homogeneity by ammonium sulfate fraction and gel filtration. The optimum pH and temperature were 5.5 and 40 °C and it was strongly resistant to SO2 and ethanol. Afterwards, the histamine degradative activity of partially purified GAPDH in actual wine environments (grape and cherry wines) was examined by incubating the enzymes in the middle, near the end and completion of malolactic fermentation, and histamine in the corresponding contaminated wines was decreased by 36.8-52.4%, 59.6-66.9% and 83.1-85.5%, respectively.

Effect of Saccharomyces cerevisiae, Torulaspora delbrueckii and malolactic fermentation on fermentation kinetics and sensory property of black raspberry wines.

Alcoholic fermentation (AF) and malolactic fermentation (MLF) both have significant influence on the production of black raspberry wine. In this study, three microbes associated with AF and MLF including S. cerevisiae, T. delbrueckii and O. oeni were used to investigate their combined effect on basic compositional, volatile and sensory property of black raspberry wine, and four fermentation trials including single S. cerevisiae inoculation plus spontaneous MLF (BSU) and controlled MLF with O. oeni (BSO), sequential culture of T. delbrueckii and S. cerevisiae plus spontaneous MLF (BTSU) and controlled MLF (BTSO) were tested and compared. Fermentation results showed MLF in BSU, BSO and BTSO were successful, with respective period of 40, 25 and 23 days, whereas a stuck MLF occurred in BTSU. Volatile compounds were determined by HS-GC-IMS method, with a total of 45 aromas identified. BTSO was distinguished by a significant higher signal intensity of many fruity esters and a lower production of several alcohols and terpenes, which was in agreement with its perception result of strong 'fruity' and slight note of 'solvent' and 'herbaceous' during quantitative descriptive analysis. On the contrary, BSU was found to reinforce the synthesis of most detected volatiles, resulting in the enhancement of both beneficial and off-flavour compounds, therefore scoring lower in the 'global aroma' descriptor. Principal component analysis showed BSU and BSO were similar in the volatile composition, whereas BTSO was quite different. Overall, BTSO had greater potential to be used in the production of black raspberry wine.

Comparison of fermentation behaviors and properties of raspberry wines by spontaneous and controlled alcoholic fermentations.

Torulaspora delbrueckii is a widely studied non-Saccharomyces yeast, described as having a positive impact on the organoleptic quality of wines, however, little is known about its impact on the production of raspberry wine. In this study, we compared combined use of S. cerevisiae/T. delbrueckii pair, i.e., in sequential inoculation (RT) and co-fermentation (RC) modes, with spontaneous fermentation (RU) and single S. cerevisiae inoculation (RS), on various properties of raspberry wine including fermentation behaviors (using yeast counts and next-generation sequencing method), basic composition (by OIV, 2019), volatile profile (using headspace-gas chromatography-ion mobility spectrometry), sensory property (by quantitative descriptive analyses) and biogenic amine levels. All the alcoholic fermentations were completed within 9 days; T. delbrueckii was inhibited by S. cerevisiae in the co-culture; and Saccharomyces and Mrakia were the most abundant species in RU. A total of 40 aromas was identified, with RT abundant in volatile esters, ketones and terpenes and others showing relatively lower intensities. During sensory evaluation, RT was characterized by 'fruity' and 'sweet' notes; RC was notable for a high 'floral' attribute; RU scored the highest in 'pungent' and RS showed intermedium intensities for most descriptors. Partial least squares regression showed the relationship between aromas and sensory descriptors. As for biogenic amine, RU contained the highest total amount and RS had the least. Overall, RT had greater potential to be used in the production of raspberry wine.

[Responses of soil intracellular and extracellular urease activities to carbon additions in chernozem].

Urease in soil, as the most important enzyme catalyzing urea hydrolysis, plays an important role in nitrogen supply of grassland ecosystems. While the effects of different carbon additions on extracellular urease in grassland soil are well understood, their effects on soil intracellular urease remain unknow. Moreover, whether the responses of intracellular and extracellular urease to carbon additions are consistent need to be clarified. With a field experiment in Hulun Buir grassland of Inner Mongolia, we investigated soil intracellular and extracellular urease activities in chernozem under four treatments, including carbon-free (C0), 250 (C250), and 500 (C500) kg C·hm-2·a-1, using glucose as carbon source. Further, we analyzed their relationships with soil properties. Carbon additions significantly increased soil intracellular urease activity and its ratio to total urease activities and total urease activity, but did not affect soil extracellular urease activities. There was significant positive correlation between soil intracellular urease activity and microbial biomass, suggesting that the increases of soil intracellular urease activities were mainly caused by the increases of microbial biomass. Results of structural equation modeling (SEM) analysis showed that carbon additions indirectly increased soil intracellular urease activities by affecting microbial biomass.