Industrial microbial strain breeding remains hindered by the inherent bottleneck of low-throughput phenotypic screening. Here, a high-throughput Raman-activated cell sorting strategy was employed to directly quantify the intracellular docosahexaenoic acid (DHA) content in single cells of the thraustochytrid Aurantiochytrium sp. SD116, eliminating the need for prolonged cultivation and lipid extraction. From a mutant library of 50,000 cells, a strain designated ABBS26, exhibiting a DHA content exceeding 65%, was isolated. Omics analysis revealed that the high DHA content of ABBS26 stems from robust lipid recycling, particularly targeting saturated fatty acids in triacylglycerol. After process optimization during fed-batch fermentation, ABBS26 achieved a final biomass of 147.3 g/L, a DHA production of 47.4 g/L, and a DHA content of 67.6% of total lipids. This work provides a promising approach for the improvement of PUFA-producing thraustochytrid strains and yields strain ABBS26, which exhibits great potential for applications in the food and pharmaceutical fields.

Microbial fatty acid-producing strains are commonly engineered to improve their performance for industrial applications. However, it is challenging to efficiently and rapidly screen target strains for engineering. This study reported an in situ analytical platform using laser ablation electrospray ionization mass spectrometry (LAESI-MS) for fast profiling of triacylglycerols in cellular lipid droplets of Aurantiochytrium sp. colonies cultured on agar plates. LAESI-MS approach allowed for the direct acquisition of a colony cell's characteristic fingerprint mass spectrum and MS/MS facilitated the identification of triacylglycerol species containing three fatty acyl groups. The fatty acid contents of colony cells were calculated based on the intensities of triacylglycerols from their characteristic fingerprint mass spectrum. A Python package called TAFA-LEMS (Triacylglycerol to Fatty Acid by LAESI-MS) was also developed to process the high-throughput MS data and extract fatty acid contents in colony cells. The results demonstrated that the LAESI-MS platform is fast, stable, and reproducible, with a data acquisition rate of ≤2 s per sampling point and ≤13.69% RSDs of the relative contents of fatty acids. In addition, LAESI-MS was successfully performed on the analysis of P. tricornutum and Y lipolytica strains. This in situ MS platform has the potential to become a common biotechnology platform for microbial strain engineering.

Microbial fuel cells (MFCs) employing Pseudomonas putida B6-2 (ATCC BAA-2545) as an exoelectrogen have been developed to harness energy from various conventional substrates, such as acetate, lactate, glucose, and fructose. Owing to its metabolic versatility, P. putida B6-2 demonstrates adaptable growth rates on diverse, cost-effective carbon sources within MFCs, exhibiting distinct energy production characteristics. Notably, the anode chamber's pH rises with carboxylates' (acetate and lactate) consumption and decreases with carbohydrates' (glucose and fructose) utilization. The MFC utilizing fructose as a substrate achieved the highest power density at 411 mW m-2. Initial analysis revealed that P. putida B6-2 forms biofilms covered with nanowires, contributing to bioelectricity generation. These microbial nanowires are likely key players in direct extracellular electron transport through physical contact. This study established a robust foundation for producing valuable compounds and bioenergy from common substrates in bioelectrochemical systems (BESs) utilizing P. putida as an exoelectrogen.

Sterols are essential lipid components for the cell membrane. In addition to their structural roles, they are critical signaling molecules that regulate metabolism, development, and homeostasis. Due to the functions of sterols being concentration.dependent, the biosynthesis of sterols is tightly controlled. Here, we reviewed the biosynthesis processes of sterols (squalene, lanosterol, ergosterol, carotenoid, and polyprenols) and analyzed the key and limited enzymes in these processes. Although various sterols are identified in nature, their basic synthesis pathways appear to be conserved. Squalene is the key intermediate in the biosynthesis of sterols, and the cyclization of squalene into lanosterol (animals and fungi) or cycloartenol (plants), producing various types of terpenoids. In addition to the synthesis processes of sterols, how to enhance sterols production was also discussed, which provides the strategy for the industrial production of sterol products.

Environmental pollution caused by aromatic compounds (ACs) has long stimulated the emergence of creative bioremediation strategies. Pseudomonas putida B6-2, an aerobic ACs degrading bacterium, successfully coupled ACs degradation with power generation in microbial fuel cells (MFCs) when a nickel-coated sponge with a three-dimensional macroporous structure served as the anode. The simultaneous removal of coexisting ACs pollutants including biphenyl (BP), carbazole (CA), and dibenzothiophene (DBT) were achieved in MFCs, accompanied by the efficient power densities of 469 mW m(-2 ), 499 mW m(-2), 541 mW m(-2), and 587 mW m(-2) for single (BP), binary (BP+DBT or BP+CA), and ternary (BP+DBT+CA) ACs fueled MFCs, respectively. Degradation dynamics analysis indicated that the degradation of BP, CA, and DBT was consistent with the pseudo-first order kinetic model in the ternary ACs fueled MFC, and BP had a higher degradation rate (k(obs )=0.0337 h(-1)) than DBT (k(obs) =0.0201 h(-1)) and CA (k(obs) =0.0149 h(-1)). It is the first time that P. putida B6-2 was discovered as an electroactive bacterium with high extracellular electron transfer (EET) capability. The EET preliminary analysis showed that P. putida B6-2 might transfer extracellular electrons by redox mediators and microbial nanowires. The mediators may play a key role in indirect EET at the start-up period of the MFCs, while the nanowires may involve in direct EET during the whole discharge process of the MFCs. The interesting findings may lay a foundation for further exploration of the EET of P. putida B6-2 and bioremediation of ACs pollutants.

Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.

Cytophaga hutchinsonii can efficiently degrade cellulose and rapidly glide over surfaces, but the underlying mechanisms remain unclear. The type IX secretion system (T9SS) is involved in protein secretion and gliding motility, which is unique to the phylum Bacteroidetes. In this study, we deleted a homologous gene of PorV (chu_3238), a shuttle protein in the T9SS. The Δ3238 mutant caused cellulolytic and gliding defects, while the porV deletion mutants in other Bacteroidetes could glide normally. Adding Ca2+ and K+ improved growth in the PY6 medium, suggesting a potential role of chu_3238 in ion uptake. A proteomic analysis showed an increase in the number of extracellular proteins in the Δ3238 mutant and a decrease in the outer membrane proteins compared to the wild type (WT). Endoglucanase activity in the Δ3238 intact cells was reduced by approximately 70% compared to that of the WT. These results indicate that the secreted proteins could not attach to the cell surface but were released into the extracellular space in the Δ3238 mutant. However, the cargo proteins accumulated in the periplasm of other reported porV deletion mutants. In addition, the homologs of the translocon SprA and a Plug protein were pulled down by co-immunoprecipitation in the 3238-FLAG strain, which are involved in protein transport in the T9SS of Flavobacterium johnsoniae. The integrity of the lipopolysaccharide (LPS) was also affected in the Δ3238 mutant, which may be the reason for the sensitivity of the cell to toxic reagents. The functional diversity of CHU_3238 suggests its important role in the T9SS of C. hutchinsonii and highlights the functional differences of PorV in the T9SS among the Bacteroidetes.

Thraustochytrid is a promising algal oil resource with the potential to meet the demand for docosahexaenoic acid (DHA). However, oils with high DHA content produced by genetic modified thraustochytrids are not accepted by the food and pharmaceutical industries in many countries. Therefore, in order to obtain non-transgenic strains with high DHA content, a two-stage adaptive laboratory evolution (ALE) strategy was applied to the thraustochytrid Aurantiochytrium sp. Heavy-ion irradiation technique was first used before the ALE to increase the genetic diversity of strains, and then two-step ALE: low temperature based ALE and ACCase inhibitor quizalofop-p-ethyl based ALE were employed in enhancing the DHA production. Using this strategy, the end-point strain E-81 with a DHA content 51% higher than that of the parental strain was obtained. The performance of E-81 strain was further analyzed by component analysis and quantitative real-time PCR. The results showed that the enhanced in lipid content was due to the up-regulated expression of key enzymes in lipid accumulation, while the increase in DHA content was due to the increased transcriptional levels of polyunsaturated fatty acid synthase. This study demonstrated a non-genetic approach to enhance lipid and DHA content in non-model industrial oleaginous strains.

Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins.IMPORTANCECytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

The crystalline region of cellulose is the main barrier to the utilization of crystalline cellulose. Cytophaga hutchinsonii actively digests the crystalline region of cellulose by an unknown mechanism. Transposon mutagenesis was done to identify a novel gene locus chu_1557 , which is required for efficient disruption of the crystalline region of cellulose, and the absence of CHU_1557 resulted in decreased glucose assimilation efficiency. The defect of the mutant in the disruption of the crystalline region of cellulose was partially retained by additional glucose or pre-culturing the mutant in a low glucose concentration medium which could improve its glucose absorption efficiency. These results suggested that extracellular glucose has important roles in the disruption of crystalline cellulose by C. hutchinsonii . Further study showed that the expression of an outer membrane protein CHU_3732 was downregulated by the absence of CHU_1557 in a low glucose concentration medium. CHU_3732 was involved in uptake of glucose and its expression was induced by a low concentration of glucose. CHU_3732 was predicted to be a porin, so we inferred that it may work as a glucose transport channel in the outer membrane. Based on these results, we deduced that CHU_1557 played a role in the process of glucose assimilation and its disruption affected the expression of other proteins related to glucose transportation such as CHU_3732, and then affected the cell growth in a low glucose concentration medium and disruption of the crystalline region of cellulose.

Cytophaga hutchinsonii can rapidly digest crystalline cellulose without free cellulases or cellulosomes. Its cell-contact cellulose degradation mechanism is unknown. In this study, the four β-glucosidase (bgl) genes in C. hutchinsonii were singly and multiply deleted, and the functions of these β-glucosidases in cellobiose and cellulose degradation were investigated. We found that the constitutively expressed BglB played a key role in cellobiose utilization, while BglA which was induced by cellobiose could partially make up for the deletion of bglB. The double deletion mutant ΔbglA/bglB lost the ability to digest cellobiose and could not thrive in cellulose medium, indicating that β-glucosidases were important for cellulose degradation. When cultured in cellulose medium, a small amount of glucose accumulated in the medium in the initial stage of growth for the wild type, while almost no glucose accumulated for ΔbglA/bglB. When supplemented with a small amount of glucose, ΔbglA/bglB started to degrade cellulose and grew in cellulose medium. We inferred that glucose might be essential for initiating cellulose degradation, and with additional glucose, C. hutchinsonii could partially utilize cellulose without β-glucosidases. We also found that there were both cellulose binding cells and free cells when cultured in cellulose. Since direct contact between C. hutchinsonii cells and cellulose is necessary for cellulose degradation, we deduced that the free cells which were convenient to explore new territory in the environment might be fed by the adherent cells which could produce cello-oligosaccharide and glucose into the environment. This study enriched our knowledge of the cellulolytic pathway of C. hutchinsonii.

ABSTRACT Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220 , encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii . A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN 3 . Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220 . Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases. IMPORTANCE Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii .

Cytophaga hutchinsonii glides rapidly over surfaces by an unknown mechanism without flagella and type IV pili and it can degrade crystalline cellulose efficiently by a novel mechanism. Tn4351 transposon mutagenesis was used to identify a new gene, CHU_1798, essential for colony spreading on agar surfaces. Further study showed that disruption of CHU_1798 caused non-spreading colonies on both soft and hard agar surfaces and individual cells were partially deficient in gliding on glass surfaces. The CHU_1798 mutant could digest cellulose as long as the cells were in direct contact with the cellulose, but it could not degrade cellulose powder buried in the agar plate. Scanning electron microscopy showed that individual mutant cells arranged irregularly on the cellulose fiber surface at an early stage of incubation, but later showed a regular parallel arrangement when there were plenty of cells and could spread along the cellulose fibers. These results suggest that CHU_1798 plays an important role in the motility of C. hutchinsonii and provide insight into the relation between cell motility and cellulose degradation.

Cytophaga hutchinsonii is an aerobic cellulolytic gliding bacterium. The mechanism of its cell motility over surfaces without flagella and type IV pili is not known. In this study, mariner-based transposon mutagenesis was used to identify a new locus CHU_1797 essential for colony spreading on both hard and soft agar surfaces through gliding. CHU_1797 encodes a putative outer membrane protein of 348 amino acids with unknown function, and proteins which have high sequence similarity to CHU_1797 were widespread in the members of the phylum Bacteroidetes. The disruption of CHU_1797 suppressed spreading toward glucose on an agar surface, but had no significant effect on cellulose degradation for cells already in contact with cellulose. SEM observation showed that the mutant cells also regularly arranged on the surface of cellulose fiber similar with that of the wild type strain. These results indicated that the colony spreading ability on agar surfaces was not required for cellulose degradation by C. hutchinsonii. This was the first study focused on the relationship between cell motility and cellulose degradation of C. hutchinsonii.