Enhancing the catalytic efficiency of M32 carboxypeptidase by semi-rational design and its applications in food taste improvement.

BACKGROUND: Carboxypeptidase is an exopeptidase that hydrolyzes amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization. RESULTS: The study has enhanced the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the specific activity of the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, was 2.2-fold higher than that of the wild type (187.9 versus 417.8 U mg-1), and the catalytic efficiency was 2.9-fold higher (110.14 versus 325.75 s-1 mmol-1). Secondly, compared to the wild type, BmeCPM32-M2 exhibited a 1.8-fold increase in half-life at 60 °C, with no significant changes in its enzymatic properties (optimal pH, optimal temperature). Finally, BmeCPM32-M2 significantly increased the umami intensity of soy protein isolate hydrolysate by 55% and reduced bitterness by 83%, indicating its potential in developing tasty protein components. CONCLUSION: Our research has revealed that the strategy based on protein sequence evolution and computational residue mutation energy led to an improved catalytic efficiency of BmeCPM32. Molecular dynamics simulations have revealed that a smaller substrate binding pocket and increased enzyme-substrate affinity are the reasons for the enhanced catalytic efficiency. Furthermore the number of hydrogen bonds and solvent and surface area may contribute to the improvement of thermostability. Finally, the de-bittering effect of BmeCPM32-M2 in soy protein isolate hydrolysate suggests its potential in developing palatable protein components. © 2024 Society of Chemical Industry.

Effect and application of proline metabolism-related gene CgMCUR1 on stress tolerance of Candida glycerinogenes and Saccharomyces cerevisiae.

AIMS: To investigate the effect of CgMCUR1 on the phenotype of Candida glycerinogenes and Saccharomyces cerevisiae. METHODS AND RESULTS: Inhibition of CgMCUR1 expression reduced acetate, H2O2, and high temperature tolerance of C. glycerinogenes. Expression of CgMCUR1 resulted in better acetic acid, H2O2, and high temperature tolerance in recombinant S. cerevisiae. Meanwhile, CgMCUR1 was able to enhance intracellular proline accumulation. The qRT-PCR analysis revealed that overexpression of CgMCUR1 affected proline metabolism in recombinant S. cerevisiae. The overexpression strain also showed reduced levels of cellular lipid peroxidation and an altered ratio of saturated fatty acid (SFA) to unsaturated fatty acid (UFA) in the cell membrane. The ethanol production of recombinant S. cerevisiae at high temperature was 30.9 g l-1, obtaining an increase of 12%, and the conversion rate was increased by 12%. In the undetoxified cellulose hydrolysate, the ethanol yield was 14.7 g l-1 at 30 h with an improvement of 18.5%, and the conversion rate was increased by 15.3%. CONCLUSIONS: Overexpression of CgMCUR1 rendered the acetic acid, H2O2, and high temperature tolerant of recombinant S. cerevisiae and enhanced the ethanol fermentation performance of recombinant S. cerevisiae under high temperature stress and in undetoxified cellulose hydrolysate by improving intracellular proline accumulation and by changing cellular physiological metabolism.

Effects of trehalose and ergosterol on pinene stress of Candida glycerinogenes.

Pinene is a commercially important monoterpene that can be prepared using engineered bacterial and yeast species; however, high pinene levels can adversely affect the stability and permeability of microbial membranes leading to significantly reduced growth yields. This study reports that the fluidities and permeabilities of cell membranes of Candida glycerinogenes decrease as pinene levels increase resulting in adverse effects on cell growth. Exposure of cells to pinene results in upregulation of the genes encoding ergosterol and trehalose whose production helps stabilize their cell membranes. Exogenous addition of ergosterol and trehalose to pinene-treated cells also reduces the fluidity and permeability of the cell membrane, whilst also reducing production of intracellular reactive oxygen species. This led to the finding that the biomass of yeast cells cultivated in shake flask systems are improved by exogenous addition of trehalose and ergosterol. Overexpression of genes that encode trehalose and ergosterol produced a recombinant C. glycerinogenes strain that was found to tolerate higher concentrations of  pinene.

Candida glycerinogenes Strains Overexpressing Transcription Factors have Improved Furfural Tolerance in Ethanol Production from Non-detoxified Cellulose Hydrolysate.

Cellulose is one of the main raw materials for production of green ethanol, but the presence of the growth inhibitor furfural in non-detoxified lignocellulosic hydrolysates often seriously affects their utilization. In a previous study, we obtained strains of Candida glycerinogenes that were tolerant to furfural, but at concentrations above 2.5 g L-1 there was a significant increase in the growth lag phase. In this work, transcription factor genes (SEF1, STB5, CAS5, and ETP1) associated with furfural tolerance were identified and employed to obtain modified strains permitting ethanol fermentation of concentrated and non-detoxified cellulose hydrolysates containing more than 2.5 g L-1 furfural. Tolerance to furfural could be increased to 4.5 g L-1 by overexpression of either STB5 or ETP1, which have different regulation patterns. Moreover, in non-detoxified and concentrated cellulose hydrolysate, overexpression of ETP1 significantly shortened the growth lag phase and ethanol fermentation time was reduced by 17-20%. In batch fermentations fed with concentrated non-detoxified lignocellulose hydrolysate, ethanol productivity and maximum ethanol concentration reached 2.4 g L-1 h-1 and 72.5 g L-1, increases of 26.1% and 6.6%, respectively. The results provided a route for the economic use of lignocellulose for chemical production.

Identification of key residues for efficient glucose transport by the hexose transporter CgHxt4 in high sugar fermentation yeast Candida glycerinogenes.

Efficient hexose transporters are essential for the development of industrial yeast strains with high fermentation performance. We previously identified a hexose transporter, CgHxt4, with excellent sugar uptake performance at ultra-high glucose concentrations (200 g/L) in the high sugar fermenting yeast C. glycerinogenes. To understand the working mechanism of this transporter, we constructed 87 mutants and examined their glucose uptake performance. The results revealed that five residues (N321, N322, F325, G426, and P427) are essential for the efficient glucose transport of CgHxt4. Subsequently, we focused our analysis on the roles of N321 and P427. Specifically, N321 and P427 are likely to play a role in glucose coordination and conformational flexibility, respectively. Our results help to expand the application potential of this transporter and provide insights into the working mechanism of yeast hexose transporter. KEY POINTS: • Five residues, transmembrane segments 7 and 10, were found to be essential for CgHxt4. • N321 and P427 are likely to play a role in glucose coordination and conformational flexibility, respectively. • Chimeric CgHxt5.4TM7 significantly enhanced the performance of CgHxt5.

Genetic engineering of an industrial yeast Candida glycerinogenes for efficient production of 2-phenylethanol.

Microbial cell factories offer an economic approach for synthesizing "natural'" aromatic flavor compounds. During their fermentation process, the inefficient synthesis pathway and product cytotoxicity are the major barriers to the high-level production. This study combined metabolic engineering and tolerance engineering strategies to maximize the valuable rose-smell 2-phenylethanol (2-PE) production in Candida glycerinogenes, a GRAS diploid industrial yeast. Firstly, 2-PE metabolic networks involved in Ehrlich pathway were stepwise rewired using metabolic engineering, including the following: (1) overexpressing L-phenylalanine permease Aap9 enhanced precursor uptake; (2) overexpressing enzymes (aminotransferase Aro9 and decarboxylase Aro10) of Ehrlich pathway increased catalytic efficiency; and (3) disrupting the formation of by-product phenylacetate catalyzed by Ald2 and Ald3 maximized the metabolic flux toward 2-PE. Then, tolerance engineering was applied by overexpression of a stress-inducible gene SLC1 in the metabolically engineered strain to further enhance 2-PE production. Combining these two approaches finally resulted in 5.0 g/L 2-PE in shake flasks, with productivity reaching 0.21 g/L/h, which were increased by 38.9% and 177% compared with those of the non-engineered strain, respectively. The 2-PE yield of this engineered strain was 0.71 g/g L-phenylalanine, corresponding to 95.9% of theoretical yield. This study provides a reference to efficiently engineering of microbial cell factories for other valuable aromatic compounds. KEY POINTS: • Metabolic engineering improved 2-PE biosynthesis. • Tolerance engineering alleviated product inhibition, contributing to 2-PE production. • The best strain produced 5.0 g/L 2-PE with 0.959 mol/mol yield and high productivity.

Transcription factor Hap5 induces gsh2 expression to enhance 2-phenylethanol tolerance and production in an industrial yeast Candida glycerinogenes.

2-Phenylethanol (2-PE) is an important flavor compound but also impairs cell growth severely, which in turn blocks its bioproduction. However, the molecular mechanism of 2-PE tolerance is unclear. In this study, a superb 2-PE stress-tolerant and producing yeast, Candida glycerinogenes, was selected to uncover the underlying mechanism of 2-PE tolerance. We discovered that Hap5 is an essential regulator to 2-PE resistance, and its induction by 2-PE stress occurs at the post-transcriptional level, rather than at the transcriptional level. Under 2-PE stress, Hap5 is activated and imported into the nucleus rapidly. Then, the nuclear Hap5 binds to the glutathione synthetase (gsh2) promoter via CCAAT box, to induce the expression of gsh2 gene. The increased gsh2 expression contributes to enhanced cellular glutathione content, and consequently alleviates ROS accumulation, lipid peroxidation, and cell membrane damage caused by 2-PE toxicity. Specifically, increasing the expression of gsh2 is effective in improving not just 2-PE tolerance (33.7% higher biomass under 29 mM 2-PE), but also 2-PE production (16.2% higher). This study extends our knowledge of 2-PE tolerance mechanism and also provides a promising strategy to improve 2-PE production.

Identification and characterization from Candida glycerinogenes of hexose transporters having high efficiency at high glucose concentrations.

During high gravity fermentation, a set of hexose transporters in yeasts plays an important role in efficient sugar transport. However, hexose transporters have been studied mainly in the Saccharomyces cerevisiae model and at low or moderate sugar concentrations. The hexose transporters are still poorly understood in the industrial glycerol producer Candida glycerinogenes, which assimilates sugar efficiently at high glucose concentration. To explore these hexose transporters, 14 candidates were identified using a hidden Markov model and characterized. Five of these functioned as hexose transporters when expressed in S. cerevisiae. In particular, CgHxt4 showed the highest efficiency of glucose transport at elevated glucose concentration among a group of transporters including Hxt1 and Hxt7 from S. cerevisiae. qRT-PCR in C. glycerinogenes revealed that transcription of CgHXT4 was induced by high glucose concentrations while fluorescence localization analysis indicated that CgHxt4 remained relatively stable on the membrane under these conditions. In addition, site-directed mutagenesis revealed that the asparagine 329 from CgHxt4, located in the YYX(T/P) conserved motif of hexose transporters, promoted an increased glucose transport. Overexpressing CgHXT4 in S. cerevisiae enhanced glucose consumption and ethanol production more effectively at high glucose concentrations than ScHXT1, the most significant native transporter from S. cerevisiae. These results indicate that CgHxt4 plays an important role in the fermentation process as a hexose transporter with strong transport activity and efficient expression regulation at high glucose concentrations.

Itaconic acid production in microorganisms.

Itaconic acid, 2-methylidenebutanedioic acid, is a precursor of polymers, chemicals, and fuels. Many fungi can synthesize itaconic acid; Aspergillus terreus and Ustilago maydis produce up to 85 and 53 g l-1, respectively. Other organisms, including Aspergillus niger and yeasts, have been engineered to produce itaconic acid. However, the titer of itaconic acid is low compared with the analogous major fermentation product, citric acid, for which the yield is > 200 g l-1. Here, we review two types of pathway for itaconic acid biosynthesis as well as recent advances by metabolic engineering strategies and process optimization to enhance itaconic acid productivity in native producers and heterologous hosts. We also propose further improvements to overcome existing problems.

Effects of xylitol dehydrogenase (XYL2) on xylose fermentation by engineered Candida glycerinogenes.

Efficient bioconversion of d-xylose into various biochemicals is critical for lignocelluloses application. Candida glycerinogenes, expressing the xylitol dehydrogenase (XYL2) gene, has the ability to produce glycerol from xylose entered into pentose phosphate pathway. In this study, we demonstrate that low expression levels of the XYL2 gene derived from Scheffersomyces stipitis in C. glycerinogenes is a major bottleneck in efficient xylose fermentation. Through a metabolic engineering approach using an integrative expression, XYL2 was identified as an overexpression target for improving xylose metabolism. Two recombinant strains with XYL2 overexpression were constructed to ferment a mixture of glucose and xylose simultaneously in batch fermentation. Compared with C. glycerinogenes (wild type), glycerol production from xylose by C. glycerinogenes (PURGAPX2) and C. glycerinogenes (PURGPDX2) was increased by 94.5% and 103.3%, respectively. It was also found that additional overexpression of XYL2 under the control of strong promoters in a xylose-fermenting strain not only reduced xylitol accumulation but also increased glycerol yields. As the expression levels of XYL2 increased, the glycerol yields gradually improved from 30.6 to 63.3 g/L, whereas the xylitol yields significantly decreased from 38.7 to 19.9 g/L. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose-fermenting strains.

Role of CgHOG1 in Stress Responses and Glycerol Overproduction of Candida glycerinogenes.

Candida glycerinogenes, the glycerol producer with excellent multi-stress tolerances, is considered to be a potential biotechnological host used in the production of glycerol and its derivatives under extreme fermentation conditions. In this study, to evaluate the multiple roles of mitogen-activated protein kinase CgHOG1, we constructed a gene disruption system in the diploid C. glycerinogenes to obtain CgHOG1 null mutant. Pseudohyphae generation of the CgHOG1 mutant under non-inducing condition indicated a repressor role in morphological transitions. Disruption of CgHOG1 resulted in increased sensitivities to osmotic, acetic acid, and oxidative stress but not involved in thermotolerance. In the CgHOG1 mutant, NaCl shock failed to stimulate the accumulation of intracellular glycerol and was fatal. In addition, the CgHOG1 mutant displayed a significant prolonged growth lag phase in YPD medium with no decrease in glycerol production, whereas the mutant cannot grow under hyperosmotic condition with no detectable glycerol in broth. These results suggested that CgHOG1 plays important roles in morphogenesis and multi-stress tolerance. The growth and glycerol overproduction under osmotic stress are heavily dependent on CgHOG1 kinase.

Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool.

Characterization of a novel cytochrome P450 from Amycolatopsis sp. CGMCC1149 for hydroxylation of lovastatin.

Wuxistatin, a novel and potent statin, is converted from lovastatin by Amycolatopsis sp. CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated to 3-hydroxymethyl lovastatin (product I) by a hydroxylase. In the current study, a novel hydroxylase gene p450lov was isolated from Amycolatopsis sp. CGMCC1149 by degenerate PCR and self-formed adaptor PCR and expressed in Escherichia coli. The gene encodes a 403-amino-acid protein with a molecular weight of 44.8 kDa and was designated as a new member of cytochrome P450 (CYP) 105 family, CYP105A44. Meanwhile, a lovastatin catalytic in vitro system was established, and an optimal hydroxylation reaction system contained 30 µM lovastatin, 600 µM NADH, 120 µM ferredoxin, 0.04 U ferredoxin-nicotinamide adenine dinucleotide phosphate reductase, and 100 µM CYP105A44 in a final volume of 200 µL Tris HCl buffer (50 mM, pH 7.4). These would be helpful for further studies on the hydroxylation of statins.

Integrative expression vectors for overexpression of xylitol dehydrogenase (XYL2) in Osmotolerant yeast, Candida glycerinogenes WL2002-5.

Yeasts are excellent hosts for the production of recombinant proteins. Candida glycerinogenes WL2002-5, an osmotolerant yeast with extremely high glycerol productivity, provides an attractive eukaryotic expression platform. The integrative vectors PURGAP-gfp and PURGPD-gfp harbouring phleomycin-resistance coding sequence and GFP coding sequence with PCgGAP, PCgGPD promoter, respectively, were constructed. The recombinant plasmid PURPpGAP-gfp with the promoter PPpGAP based on the sequence of Pichia pastoris GAPDH gene and the plasmid PURScGAP-gfp with the promoter PScGAP from Saccharomyces cerevisiae were constructed. After transformation, the copy number of gfp gene, which determined using fluorescent quantitative real-time polymerase chain reaction (FQ-RTPCR) in genome of C. glycerinogenes is 1. Expressions of gfp at different levels were conducted using different promoters by osmotic stress containing NaCl or glucose for the recombinant strains. In this study, C. glycerinogenes WL2002-5, expressing xylitol dehydrogenase (XYL2) gene from Pichia stipitis, has the ability to produce glycerol from xylose entered into pentose phosphate pathway. Two recombinant strains of PURGAPX, PURGPDX with XYL2 overexpression were constructed to ferment a mixture of glucose and xylose simultaneously in batch fermentation. Compared to C. glycerinogenes WL2002-5 strain, glycerol production from xylose in strains PURGAPX, PURGPDX were increased by 95.9 and 121.1 %, respectively.

[New osmo-regulational promoters in the industrial yeast].

OBJECTIVE: The work was aimed at selecting osmo-regulated prompters possessing excellent performance for further research of the industrial yeast Candida glycerinogenes. METHODS: Promoters PCgPGI, PCgTPI, PCgZWF, PCgSTL1, PCgSTL2 and PCgSTL3 were amplified by PCR and their bioinformatics analysis of stress response elements (STREs) were conducted. We constructed integrative plasmids containing 5.8S rDNA, a fluorescence protein gene gfp and a promoter PCgPGI, PCgTPI, PCgZWF, PCgSTL1, PCgSTL2 or PCgSTL3. The promoters' activities and osmo-regulations were compared according to the results of fluorescence and qRT-PCR. RESULTS: PCgSTL3 had more STREs, higher transcription level, lager gfp expression and it was more sensitive to stress. CONCLUSION: PCgSTL3 is an excellent induced promoter responding to hyperosmotic stress. Controlled expression of target genes can be realized using PCgSTL3 in the industrial yeast.

Identification of a novel HOG1 homologue from an industrial glycerol producer Candida glycerinogenes.

Candida glycerinogenes, a glycerol production industrial strain with hyperosmo-adaptation can grow well in 15 % (w/v) NaCl or 55 % (w/v) glucose. To understand the osmo-adaptation mechanism in C. glycerinogenes, the mitogen-activated protein kinase HOG1 gene (CgHOG1), which plays an essential role in the yeast hyperosmotic response, was isolated by degenerate PCR and SEFA-Formed Adaptor PCR. The CgHOG1 gene was then transformed in Saccharomyces cerevisiae hog1Δ null mutant, which restored the recombination S. cerevisiae to the wild-type phenotype with osmo-adaptation. To further clarify the function of CgHOG1, the phosphorylation of CgHOG1 and transcription of the glycerol-3-phosphate dehydrogenase gene (GPD1) of the CgHOG1-harbouring S. cerevisiae mutant was detected, and found to be similar to that of wild-type S. cerevisiae. In addition, the recombination S. cerevisiae with CgHOG1 gene significantly accumulated intracellular glycerol when stressed with NaCl.

Gene Editing of Candida glycerinogenes by Designed Toxin-Antitoxin Cassette.

Candida glycerinogenes is an industrial yeast with excellent multistress resistance. However, due to the diploid genome and the lack of meiosis and screening markers, its molecular genetic operation is limited. Here, a gene editing system using the toxin-antitoxin pair relBE from the type II toxin-antitoxin system in Escherichia coli as a screening marker was constructed. The RelBE complex can specifically and effectively regulate cell growth and arrest through a conditionally controlled toxin RelE switch, thereby achieving the selection of positive recombinants. The constructed editing system achieved precise gene deletion, replacement, insertion, and gene episomal expression in C. glycerinogenes. Compared with the traditional amino acid deficiency complementation editing system, this editing system produced higher biomass and the gene deletion efficiency was increased by 3.5 times. Using this system, the production of 2-phenylethanol by C. glycerinogenes was increased by 11.5-13.5% through metabolic engineering and tolerance engineering strategies. These results suggest that the stable gene editing system based on toxin-antitoxin pairs can be used for gene editing of C. glycerinogenes to modify metabolic pathways and promote industrial applications. Therefore, the constructed gene editing system is expected to provide a promising strategy for polyploid industrial microorganisms lacking gene manipulation methods.

Regulatory mechanisms and cell membrane properties of Candida glycerinogenes differ under 2-phenylethanol addition or fermentation conditions.

The biosynthesis of 2-phenylethanol (2-PE) at high yields and titers is often limited by its toxicity. In this study, we describe the molecular mechanisms of 2-PE tolerance in the multi-stress tolerant industrial yeast, Candida glycerinogenes. They were different under 2-PE addition or fermentation conditions. After extracellular addition of 2-PE, C. glycerinogenes cells became rounder and bigger, which reduced specific surface area. However, during 2-PE fermentation C. glycerinogenes cells were smaller, which increased specific surface area. Other differences in the tolerance mechanisms were studied by analyzing the composition and molecular parameters of the cell membrane. Extracellular 2-PE stress resulted in down-regulation of transcriptional expression of unsaturated fatty acid synthesis genes. This raised the proportion of saturated fatty acids in the cell membrane, which increased rigidity of the cell membrane and reduced 2-PE entry to the cell. However, intracellular 2-PE stress resulted in up-regulation of transcriptional expression of unsaturated fatty acid synthesis genes, and increased the proportion of unsaturated fatty acids in the cell membrane; this in turn enhanced flexibility of the cell membrane which accelerated efflux of 2-PE. These contrasting mechanisms are mediated by transcriptional factors Hog1 and Swi5. Under 2-PE addition, C. glycerinogenes activated Hog1 and repressed Swi5 to upregulate erg5 and erg4 expression, which increased cell membrane rigidity and resisted 2-PE import. During 2-PE fermentation, C. glycerinogenes activated Hog1 and repressed Swi5 to upregulate 2-PE transporter proteins cdr1 and Acyl-CoA desaturase 1 ole1 to increase 2-PE export, thus reducing 2-PE intracellular toxicity. The results provide new insights into 2-PE tolerance mechanisms at the cell membrane level and suggest a novel strategy to improve 2-PE production by engineering anti-stress genes.

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.

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.