Candida glycerinogenes-Promoted α‑Pinene and Squalene Co-production Strategy Based on α‑Pinene Stress

α-Pinene is a naturally occurring monoterpene, which is widely used in fragrances, cosmetics, and foods. Due to the high cellular toxicity of α-pinene, this work considered the application of Candida glycerinogenes, an effective industrial strain with high resistance, in α-pinene synthesis. It was found that α-pinene-induced stress resulted in an intracellular accumulation of reactive oxygen species with an increased formation of squalene as a cytoprotective compound. As squalene is a downstream product in the mevalonate (MVA) pathway for α-pinene synthesis, a strategy based on the promotion of α-pinene and squalene co-production under α-pinene stress is proposed. By introducing the α-pinene synthesis pathway and enhancing the MVA pathway, the production of both α-pinene and squalene is increased. We have demonstrated that intracellular synthesis of α-pinene is effective in promoting squalene synthesis. The generation of intercellular reactive oxygen that accompanies α-pinene synthesis promotes squalene synthesis with a resultant cellular protection and upregulation of MVA pathway genes that facilitate α-pinene production. In addition, we have overexpressed phosphatase and introduced NPP as a substrate to synthesize α-pinene, where co-dependent fermentation yielded 208 mg/L squalene and 12.8 mg/L α-pinene. This work establishes a viable strategy to promote terpene-co-dependent fermentation based on stress

Gene Editing of <i>Candida glycerinogenes</i> 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

Data_Sheet_1_Development of a co-culture system for green production of caffeic acid from sugarcane bagasse hydrolysate.PDF

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.</p

Balancing Pyruvate Node Based on a Dual-Layered Dynamic Regulation System to Improve the Biosynthesis of Caffeic Acid in Candida glycerinogenes

Caffeic acid is a phenolic acid compound widely applied in the food and pharmaceutical fields. Currently, one of the reasons for the low yield of caffeic acid biosynthesis is that the carbon flow enters mainly into the TCA cycle via pyruvate, which leads to low concentrations of erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP), the precursors of caffeic acid synthesis. Here, we developed a growth-coupled dual-layered dynamic regulation system. This system controls intracellular pyruvate supply in real time by responding to intracellular pyruvate and p-coumaric acid concentrations, autonomously coordinates pathway gene expression, and redirects carbon metabolism to balance cell growth and caffeic acid synthesis. Finally, our constructed engineered strain based on the dual-layered dynamic regulation system achieved a caffeic acid titer of 559.7 mg/L in a 5 L bioreactor. Thus, this study demonstrated the efficiency and potential of this system in boosting the yield of aromatic compounds

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

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

Chemical constituents, antibacterial activity and mechanism of <i>Paeonia suffruticosa</i> Andr. buds extract against <i>Staphylococcus aureus</i> and <i>Escherichia coli</i> O157:H7

Sixteen chemical constituents of Paeonia suffruticosa Andr. buds extract (PSABE) were identified by UHPLC-PDA-Q/TOF-MS, belonging to phenolic acids, flavonoids, monoterpene glycosides and gallotannins. PSABE exhibited significant antibacterial activity against six tested microorganisms. Particularly, it showed the most efficient antibacterial effect against Staphylococcus aureus and Escherichia coli O157:H7, which the minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC) both were 1.56 mg/mL and 6.25 mg/mL, respectively. The results showed that PSABE induced obvious alterations in membrane fatty acid composition of S. aureus and E. coli O157:H7, such as the decrease of unsaturated fatty acids, leading to the reduce of membrane fluidity. Membrane integrity was destroyed and cell morphology was obviously changed with PSABE. Furthermore, the transcription level of virulence factors was inhibited in the presence of PSABE. These results indicated that PSABE mainly exerted antibacterial effect by damaging cell membrane and inhibiting transcription level of virulence factors.</p

Metabolic Engineering of Candida glycerinogenes for Sustainable Production of Geraniol

Geraniol is a class of natural products that are widely used in the aroma industry due to their unique aroma. Here, to achieve the synthesis of geraniol and alleviate the intense competition from the yeast ergosterol pathway, a transcription factor-mediated ergosterol feedback system was developed in this study to autonomously regulate ergosterol metabolism and redirect carbon flux to geraniol synthesis. In addition, the modification of ergosterol-responsive promoters, the optimization of transcription factor expression intensity, and stepwise metabolic engineering resulted in a geraniol titer of 531.7 mg L–1. For sustainable production of geraniol, we constructed a xylose assimilation pathway in Candida glycerinogenes (C. glycerinogenes). Then, the xylose metabolic capacity was ameliorated and the growth of the engineered strain was rescued by activating the pentose phosphate (PP) pathway. Finally, we obtained 1091.6, 862.4, and 921.8 mg L–1 of geraniol in a 5 L bioreactor by using pure glucose, simulated wheat straw hydrolysates, and simulated sugarcane bagasse hydrolysates, with yields of 47.5, 57.9, and 59.1 mg g–1 DCW, respectively. Our study demonstrated that C. glycerinogenes has the potential to produce geraniol from lignocellulosic biomass, providing a powerful tool for the sustainable synthesis of other valuable monoterpenes

Production of Caffeic Acid with Co-fermentation of Xylose and Glucose by Multi-modular Engineering in Candida glycerinogenes

Caffeic acid (CA), a natural phenolic compound, has important medicinal value and market potential. In this study, we report a metabolic engineering strategy for the biosynthesis of CA in Candida glycerinogenes using xylose and glucose. The availability of precursors was increased by optimization of the shikimate (SA) pathway and the aromatic amino acid pathway. Subsequently, the carbon flux into the SA pathway was maximized by introducing a xylose metabolic pathway and optimizing the xylose assimilation pathway. Eventually, a high yielding strain CG19 was obtained, which reached a yield of 4.61 mg/g CA from mixed sugar, which was 1.2-fold higher than that of glucose. The CA titer in the 5 L bioreactor reached 431.45 mg/L with a yield of 8.63 mg/g of mixed sugar. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of CA. This is the first report to produce CA in C. glycerinogenes with xylose and glucose as carbon sources, which developed a promising strategy for the efficient production of high-value aromatic compounds

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