Toxicants improve glycerol production in the fermentation of undetoxified hydrolysate by Candida glycerinogenes.

OBJECTIVES: Toxicants inhibit microbial fermentation and reduce product titres. This work investigated the glycerol production characteristics of Candida glycerinogenes in highly toxic unwashed undetoxified hydrolysate and provided new ideas for high glycerol production from hydrolysates. RESULTS: The unwashed hydrolysate contains higher concentrations of toxicants, such as furfural, acetic acid, phenols and NaCl than the washed alkali-treated bagasse hydrolysate. C. glycerinogenes fermented unwashed undetoxified hydrolysate yielded 36.1 g/L glycerol, 15.8% higher than the washed hydrolysate, suggesting that the toxicants stimulated glycerol synthesis. qRT-PCR analysis showed that toxicants of unwashed undetoxified hydrolysates greatly up-regulated the transcript levels of the genes GPD1, HXT4 and MSN4 et al. Overexpressing the above genes increased glycerol production by 27.9% to 46.1 g/L. And it was further increased by 8.8% to 50.1 g/L in a 5 L bioreactor. CONCLUSIONS: This result proves that toxicants in lignocellulosic hydrolysates can increase the titre of microbial glycerol production.

Effect of Goji Berry extract on cell viability of Lactiplantibacillus plantarum M5 microcapsules during in vitro gastrointestinal digestion.

Lactiplantibacillus plantarum M5 and Goji Berry extract were co-microencapsulated to maintain the activity of cells during gastrointestinal digestion, and the mechanism by which they could maintain high activity was investigated. The results showed that the microcapsules with 3% Goji Berry extract(A-GE-3) had the largest encapsulation efficiency(EE) of 92.41 ± 0.58%. SEM showed that the structure of A-GE-3 microcapsules were smoother and denser. Cell viability in A-GE-3 microcapsules remained at 7.17 log10 CFU/g after gastrointestinal digestion. Meanwhile, during the gastrointestinal digestion with 3% Goji Berry extract, cell membrane damage detected by fluorescent probes propidium iodide(PI) and 1.1-N-phenylnaphthylamine(NPN) was significantly reduced; the contents of arginine, glutamic acid and oleic acid in cell membrane were increased, which helped to maintain the dynamic balance of intracellular pH and regulated cell membrane fluidity in response to gastrointestinal environment. This study demonstrated the potential of Goji Berry extract as a probiotic protector in gastrointestinal digestion.

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.

Development of a co-culture system for green production of caffeic acid from sugarcane bagasse hydrolysate.

Caffeic acid (CA) is a phenolic acid compound widely used in pharmaceutical and food applications. However, the efficient synthesis of CA is usually limited by the resources of individual microbial platforms. Here, a cross-kingdom microbial consortium was developed to synthesize CA from sugarcane bagasse hydrolysate using Escherichia coli and Candida glycerinogenes as chassis. In the upstream E. coli module, shikimate accumulation was improved by intensifying the shikimate synthesis pathway and blocking shikimate metabolism to provide precursors for the downstream CA synthesis module. In the downstream C. glycerinogenes module, conversion of p-coumaric acid to CA was improved by increasing the supply of the cytoplasmic cofactor FAD(H2). Further, overexpression of ABC transporter-related genes promoted efflux of CA and enhanced strain resistance to CA, significantly increasing CA titer from 103.8 mg/L to 346.5 mg/L. Subsequently, optimization of the inoculation ratio of strains SA-Ec4 and CA-Cg27 in this cross-kingdom microbial consortium resulted in an increase in CA titer to 871.9 mg/L, which was 151.6% higher compared to the monoculture strain CA-Cg27. Ultimately, 2311.6 and 1943.2 mg/L of CA were obtained by optimization of the co-culture system in a 5 L bioreactor using mixed sugar and sugarcane bagasse hydrolysate, respectively, with 17.2-fold and 14.6-fold enhancement compared to the starting strain. The cross-kingdom microbial consortium developed in this study provides a reference for the production of other aromatic compounds from inexpensive raw materials.

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.

Spatiotemporal Regulation and Transport Engineering for Sustainable Production of Geraniol in Candida glycerinogenes.

Geraniol is an attractive natural monoterpene with significant industrial and commercial value in the fields of pharmaceuticals, condiments, cosmetics, and bioenergy. The biosynthesis of monoterpenes suffers from the availability of key intermediates and enzyme-to-substrate accessibility. Here, we addressed these challenges in Candida glycerinogenes by a plasma membrane-anchoring strategy and achieved sustainable biosynthesis of geraniol using bagasse hydrolysate as substrate. On this basis, a remarkable 2.4-fold improvement in geraniol titer was achieved by combining spatial and temporal modulation strategies. In addition, enhanced geraniol transport and modulation of membrane lipid-associated metabolism effectively promoted the exocytosis of toxic monoterpenes, significantly improved the resistance of the engineered strain to monoterpenes and improved the growth of the strains, resulting in geraniol yield up to 1207.4 mg L-1 at shake flask level. Finally, 1835.2 mg L-1 geraniol was obtained in a 5 L bioreactor using undetoxified bagasse hydrolysate. Overall, our study has provided valuable insights into the plasma membrane engineering of C. glycerinogenes for the sustainable and green production of valuable compounds.

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.

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.

Fine-Tuned Gene Expression Elements from Hybrid Promoter Libraries in Pichia pastoris.

As a desirable microbial cell factory, Pichia pastoris has garnered extensive utilization in metabolic engineering. Nevertheless, the lack of fine-tuned gene expression components has significantly constrained the potential scope of applications. Here, a gradient strength promoter library was constructed by random hybridization and high-throughput screening. The hybrid promoter, phy47, performed best with 2.93-fold higher GFP expression levels than GAP. The broad applicability of the novel hybrid promoter variants in biotechnological production was further validated in the biosynthesis of pinene and rHuPH20 with higher titers. The upstream regulatory sequences (UASE and URSD) were identified and applied to promoters GAP and ENO1, resulting in a 34 and 43% increase and an 18 and 37% decrease in the expression level, respectively. Yeast one-hybrid analysis showed that transcription factor HAP2 activates the hybrid promoter through a direct interaction with the crucial regulatory region UASH. Furthermore, a short segment of tunable activation sequence (20 bp) was also screened, and artificial promoters were constructed in tandem with the addition of regulatory sequence, resulting in a 61% expansion of the expression range. This study provides a molecular tool and regulatory elements for further synthetic biology research in P. pastoris.