Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering.

Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel "PEACES" system by (1) expressing the NAD + kinase gene ppnk from Clostridium glutamicum and the NADP + -dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.

Coordinating caffeic acid and salvianic acid A pathways for efficient production of rosmarinic acid in Escherichia coli.

Rosmarinic acid is a natural hydroxycinnamic acid ester used widely in the food and pharmaceutical industries. Although many attempts have been made to screen rate-limiting enzymes and optimize modules through co-culture fermentation, the titer of rosmarinic acid remains at the microgram level by microorganisms. A de novo biosynthetic pathway for rosmarinic acid was constructed based on caffeic acid synthesis modules in Escherichia coli. Knockout of competing pathways increased the titer of rosmarinic acid and reduced the synthesis of rosmarinic acid analogues. An L-amino acid deaminase was introduced to balance metabolic flux between the synthesis of caffeic acid and salvianic acid A. The ratio of FADH2/FAD was maintained via the coordination of deaminase and HpaBC, which is responsible for caffeic acid synthesis. Knockout of menI, encoding an endogenous thioesterase, increased the stability of caffeoyl-CoA. The final strain produced 5780.6 mg/L rosmarinic acid in fed-batch fermentation, the highest yet reported for microbial production. The strategies applied in this study lay a foundation for the synthesis of other caffeic acid and rosmarinic acid derivatives.

Engineering Gluconobacter cerinus CGMCC 1.110 for direct 2-keto-L-gulonic acid production.

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9 g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9 g/L.

CRISPR/Cpf1-FOKI-induced gene editing in Gluconobacter oxydans.

Gluconobacter oxydans is an important Gram-negative industrial microorganism that produces vitamin C and other products due to its efficient membrane-bound dehydrogenase system. Its incomplete oxidation system has many crucial industrial applications. However, it also leads to slow growth and low biomass, requiring further metabolic modification for balancing the cell growth and incomplete oxidation process. As a non-model strain, G. oxydans lacks efficient genome editing tools and cannot perform rapid multi-gene editing and complex metabolic network regulation. In the last 15 years, our laboratory attempted to deploy multiple CRISPR/Cas systems in different G. oxydans strains and found none of them as functional. In this study, Cpf1-based or dCpf1-based CRISPRi was constructed to explore the targeted binding ability of Cpf1, while Cpf1-FokI was deployed to study its nuclease activity. A study on Cpf1 found that the CRISPR/Cpf1 system could locate the target genes in G. oxydans but lacked the nuclease cleavage activity. Therefore, the CRISPR/Cpf1-FokI system based on FokI nuclease was constructed. Single-gene knockout with efficiency up to 100% and double-gene iterative editing were achieved in G. oxydans. Using this system, AcrVA6, the anti-CRISPR protein of G. oxydans was discovered for the first time, and efficient genome editing was realized.

Combining Metabolic Engineering and Lipid Droplet Assembly to Achieve Campesterol Overproduction in Saccharomyces cerevisiae.

Campesterol is a kind of important functional food additive. Therefore, stable and efficient campesterol biosynthesis is significant. Herein, we first knocked out the sterol 22-desaturase gene in Saccharomyces cerevisiae and expressed sterol Δ7-reductase from Pangasianodon hypophthalmus, obtaining a strain that produced 6.6 mg/L campesterol. Then, the modular expression of campesterol synthesis enzymes was performed, and a campesterol titer of 88.3 mg/L was achieved. Because campesterol is a lipid-soluble macromolecule, we promoted lipid droplet formation by exploring regulatory factors, and campesterol production was improved to 169.20 mg/L. Next, triacylglycerol lipase was used to achieve compartment campesterol synthesis. After enhancing the expression of sterol Δ7-reductase and screening cations, the campesterol titer reached 438.28 mg/L in a shake flask and 1.44 g/L in a 5 L bioreactor, which represents the highest campesterol titer reported to date. Metabolic regulation combined with lipid droplet engineering may be useful for the synthesis of other steroids as well.

High efficiency production of 5-hydroxyectoine using metabolically engineered Escherichia coli.

The 5-hydroxyectoine is a natural protective agent with long-lasting moisturising and radiation resistance properties. It can be naturally synthesized by some extremophiles using the "bacterial milking" process, but this can corrode bioreactors and downstream purification may cause environmental pollution. In this study, an engineered Escherichia coli (E. coli) strain was constructed for the 5-hydroxyectoine production. First, three ectoine hydroxylases were characterised and the enzyme from Halomonas elongata was the most effective. The L-2,4-diaminobutyrate transaminase mutant was introduced into the engineered strain, which could accumulate 2.8 g/L 5-hydroxyectoine in shake flasks. By activating the glyoxylate cycle and balancing the α-ketoglutarate distribution, the 5-hydroxyectoine titer was further increased to 3.4 g/L. Finally, the optimized strain synthesized 58 g/L 5-hydroxyectoine via a semi-continuous feeding process in a NaCl-free medium. Overall, this study reported the highest titer of 5-hydroxyectoine synthesized by E. coli and established a low-salt fermentation process through the aforementioned efforts.

Engineering Gluconbacter oxydans with efficient co-utilization of glucose and sorbitol for one-step biosynthesis of 2-keto-L-gulonic.

As the highest-demand vitamin, the development of a one-step vitamin C synthesis process has been slow for a long time. In previous research, a Gluconobacter oxydans strain (GKLG9) was constructed that can directly synthesize 2-keto-L-gulonic acid (2-KLG) from glucose, but carbon source utilization remained low. Therefore, this study first identified the gene 4kas (4-keto-D-arabate synthase) to reduce the loss of extracellular carbon and inhibit the browning of fermentation broth. Then, promoter engineering was conducted to enhance the intracellular glucose transport pathway and concentrate intracellular glucose metabolism on the pentose phosphate pathway to provide more reducing power. Finally, by introducing the D-sorbitol pathway, the titer of 2-KLG was increased to 38.6 g/L within 60 h in a 5-L bioreactor, with a glucose-to-2-KLG conversion rate of about 46 %. This study is an important step in the development of single-bacterial one-step fermentation to produce 2-KLG.

Combined engineering of l-sorbose dehydrogenase and fermentation optimization to increase 2-keto-l-gulonic acid production in Escherichia coli.

One-step fermentation to produce 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, is a long-term goal. Improvement of the enzyme's activity through engineering could benefit 2-KLG production. This study aimed to conduct a semi-rational design of l-sorbose dehydrogenase (SDH) through structure-directed, to screen mutants that could enhance the 2-KLG titer. First, the predicted structure of SDH was obtained using AlphaFold2. The key mutation sites in the substrate pocket were identified by Ala scanning. Subsequently, the mutant V336I/V368A was obtained by iterative saturation mutagenesis, which increased the yield of 2-KLG 1.9-fold. Finally, 5.03 g/L of 2-KLG was obtained by a two-stage temperature control fermentation method, and the conversion rate was 50%. Furthermore, experiments showed that knockdown of the l-sorbose-associated phosphotransferase system delays 2-KLG production. The results show that the production of 2-KLG was effectively increased through a combination of SDH engineering and fermentation optimization.

Synergetic engineering of Escherichia coli for efficient production of l-tyrosine.

l-Tyrosine, an aromatic non-essential amino acid, is the raw material for many important chemical products, including levodopa, resveratrol, and hydroxytyrosol. It is widely used in the food, drug, and chemical industries. There are many studies on the synthesis of l-tyrosine by microorganisms, however, the low titer of l-tyrosine limited the industrial large-scale production. In order to enhance l-tyrosine production in Escherichia coli, the expression of key enzymes in the shikimate pathway was up- or down-regulated. The l-tyrosine transport system and the acetic acid biosynthesis pathway were modified to further enhance l-tyrosine production. In addition, the phosphoketolase pathway was introduced in combination with cofactor engineering to redirect carbon flux to the shikimate pathway. Finally, after adaptive laboratory evolution to low pH an optimal strain was obtained. The strain can produce 92.5 g/L of l-tyrosine in a 5-L fermenter in 62 h, with a yield of 0.266 g/g glucose.

Enhancing Glycosylation of Flavonoids by Engineering the Uridine Diphosphate Glucose Supply in Escherichia coli.

Glycosylation can enhance the solubility and stability of flavonoids. The main limitation of the glycosylation process is low intracellular uridine diphosphate glucose (UDPG) availability. This study aimed to create a glycosylation platform strain in Escherichia coli BL21(DE3) by multiple metabolic engineering of the UDPG supply. Glycosyltransferase TcCGT1 was introduced to synthesize vitexin and orientin from apigenin and luteolin, respectively. To further expand this glycosylation platform strain, not only were UDP rhamnose and UDP galactose synthesis pathways constructed, but rhamnosyltransferase (GtfC) and galactosyltransferase (PhUGT) were also introduced, respectively. In a 5 L bioreactor with apigenin, luteolin, kaempferol, and quercetin as glycosyl acceptors, vitexin, orientin, afzelin, quercitrin, hyperoside, and trifolin glycosylation products reached 17.2, 36.5, 5.2, 14.1, 6.4, and 11.4 g/L, respectively, the highest titers reported to date for all. The platform strain has great potential for large-scale production of glycosylated flavonoids.

Production of Natural Pigments Using Microorganisms.

Pigments are involved in many aspects of human life, including food, cosmetics, and textiles. At present, the pigment market is mainly occupied by synthetic pigments. However, synthetic pigments have gradually presented safety and environmental problems. Therefore, humans have begun to focus on the use of natural pigments. In contrast to the extraction of pigments from plants and animals, the production of natural pigments by microbial fermentation is not affected by season and region. This review highlights recent advances in microbial synthesis of natural pigments, classifying them into various groups, including flavonoids, isoprenoids, porphyrins, N-heterocyclics, polyketides, and others. The biosynthetic pathways for each group are elucidated along with the latest progress made in enhancing production efficiency for both natural and non-natural microorganisms. Additionally, challenges associated with economically producing natural pigments using microorganisms are also discussed. This review provides a reference for researchers to replace synthetic pigments with natural pigments.

Efficient production of 2-keto-L-gulonic acid from D-glucose in Gluconobacter oxydans ATCC9937 by mining key enzyme and transporter.

Direct production of 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) from D-glucose through 2,5-diketo-D-gluconic acid (2,5-DKG) is a promising alternative route. To explore the pathway of producing 2-KLG from D-glucose, Gluconobacter oxydans ATCC9937 was selected as a chassis strain. It was found that the chassis strain naturally has the ability to synthesize 2-KLG from D-glucose, and a new 2,5-DKG reductase (DKGR) was found on its genome. Several major issues limiting production were identified, including the insufficient catalytic capacity of DKGR, poor transmembrane movement of 2,5-DKG and imbalanced D-glucose consumption flux inside and outside of the host strain cells. By identifying novel DKGR and 2,5-DKG transporter, the whole 2-KLG biosynthesis pathway was systematically enhanced by balancing intracellular and extracellular D-glucose metabolic flux. The engineered strain produced 30.5 g/L 2-KLG with a conversion ratio of 39.0%. The results pave the way for a more economical large-scale fermentation process for vitamin C.

Structural Insight into the Catalytic Mechanisms of an L-Sorbosone Dehydrogenase.

L-Sorbosone dehydrogenase (SNDH) is a key enzyme involved in the biosynthesis of 2-keto-L-gulonic acid , which is a direct precursor for the industrial scale production of vitamin C. Elucidating the structure and the catalytic mechanism is essential for improving SNDH performance. By solving the crystal structures of SNDH from Gluconobacter oxydans WSH-004, a reversible disulfide bond between Cys295 and the catalytic Cys296 residues is discovered. It allowed SNDH to switch between oxidation and reduction states, resulting in opening or closing the substrate pocket. Moreover, the Cys296 is found to affect the NADP+ binding pose with SNDH. Combining the in vitro biochemical and site-directed mutagenesis studies, the redox-based dynamic regulation and the catalytic mechanisms of SNDH are proposed. Moreover, the mutants with enhanced activity are obtained by extending substrate channels. This study not only elucidates the physiological control mechanism of the dehydrogenase, but also provides a theoretical basis for engineering similar enzymes.

Preliminary Experience with Continuous Submucosal Anastomosis in Small-Diameter Hepaticojejunostomy during Single-Port Laparoscopic Choledochal Cyst Surgery in Children.

PURPOSE: Hepaticojejunostomy anastomosis (HJA) is the most challenging aspect in single-port laparoscopic choledochal cystectomy and Roux-en-Y hepaticojejunostomy (SPCH) in children, especially in small-diameter anastomoses (diameters less than 5 mm), which are more susceptible to anastomotic stricture. We developed the continuous submucosal technique for HJA (CS-HJA) to lessen postoperative complications. The purpose of this study is to introduce our preliminary experiences with CS-HJA. METHODS: We retrospectively analyzed all available clinical data of children who underwent SPCH surgery between March 2020 and October 2022. We operated with CS-HJA on 10 children who were diagnosed with small-diameter hepaticojejunostomy (diameter less than 5 mm). Data collection mainly included demographic information, imaging data, perioperative details, and postoperative outcomes. Ten patients were included in this study. The average patient age was 55.2 months; the age range was 3 to 120 months, and the average weight was 11.6 kg; male-female ratio was 1:9. The choledocho had fusiform dilatation in five cases and cystic dilatation in five cases. There was no dilatation of the left and right hepatic ducts or intrahepatic bile ducts in all patients. All patients had no dilatation of the left and right hepatic ducts or intrahepatic bile ducts. All patients underwent a single-port laparoscopic bile-intestinal anastomosis using a submucosal jejunal anastomosis technique. Analysis of the duration of the bile-intestinal anastomosis, the length of the child's stay in the hospital after surgery, the intraoperative complications, and the postoperative complications was performed. RESULTS: All the 10 patients underwent successful SPCH by CS-HJA technique. The average length of time for hepaticojejunostomy ranged from 22 to 40 minutes, and the postoperative hospital stay was 5.2 to 9.2 days. There were no instances of bile leakage following the operation. At 17 to 30 months of follow-up, there was no abdominal pain or jaundice, and the reexamination of transaminases, bilirubin, and amylase were normal. Ultrasonography showed no bile duct stricture or dilated bile ducts, and the incision is elegant, and the families of the patients were satisfied. CONCLUSION: In SPCH surgery in children, the CS-HJA technique is safe and feasible for small-diameter hepaticojejunostomy.

Dehydrogenases of acetic acid bacteria.

Acetic acid bacteria (AAB) are a group of bacteria that can oxidize many substrates such as alcohols and sugar alcohols and play important roles in industrial biotechnology. A majority of industrial processes that involve AAB are related to their dehydrogenases, including PQQ/FAD-dependent membrane-bound dehydrogenases and NAD(P)+-dependent cytoplasmic dehydrogenases. These cofactor-dependent dehydrogenases must effectively regenerate their cofactors in order to function continuously. For PQQ, FAD and NAD(P)+ alike, regeneration is directly or indirectly related to the electron transport chain (ETC) of AAB, which plays an important role in energy generation for aerobic cell growth. Furthermore, in changeable natural habitats, ETC components of AAB can be regulated so that the bacteria survive in different environments. Herein, the progressive cascade in an application of AAB, including key dehydrogenases involved in the application, regeneration of dehydrogenase cofactors, ETC coupling with cofactor regeneration and ETC regulation, is systematically reviewed and discussed. As they have great application value, a deep understanding of the mechanisms through which AAB function will not only promote their utilization and development but also provide a reference for engineering of other industrial strains.

Combined evolutionary and metabolic engineering improve 2-keto-L-gulonic acid production in Gluconobacter oxydans WSH-004.

The direct fermentation of the precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG), has been a long-pursued goal. Previously, a strain of Gluconobacter oxydans WSH-004 was isolated that produced 2.5 g/L 2-KLG, and through adaptive evolution engineering, the strain G. oxydans MMC3 could tolerate 300 g/L D-sorbitol. This study verified that the sndh-sdh gene cluster encoded two key dehydrogenases for the 2-KLG biosynthesis pathway in this strain. Then G. oxydans MMC3 further evolved through adaptive evolution to G. oxydans 2-KLG5, which can tolerate high concentrations of D-sorbitol and 2-KLG. Finally, by increasing the gene expression levels of the sndh-sdh and terminal oxidase cyoBACD in G. oxydans 2-KLG5, the 2-KLG accumulation in the 5-L fermenter increased to 45.14 g/L by batch fermentation. The results showed that combined evolutionary and metabolic engineering efficiently improved the direct production of 2-KLG from D-sorbitol in G. oxydans.

Cereal grain 3D point cloud analysis method for shape extraction and filled/unfilled grain identification based on structured light imaging.

Cereals are the main food for mankind. The grain shape extraction and filled/unfilled grain recognition are meaningful for crop breeding and genetic analysis. The conventional measuring method is mainly manual, which is inefficient, labor-intensive and subjective. Therefore, a novel method was proposed to extract the phenotypic traits of cereal grains based on point clouds. First, a structured light scanner was used to obtain the grains point cloud data. Then, the single grain segmentation was accomplished by image preprocessing, plane fitting, region growth clustering. The length, width, thickness, surface area and volume was calculated by the specified analysis algorithms for grain point cloud. To demonstrate this method, experimental materials included rice, wheat and corn were tested. Compared with manual measurement results, the average measurement error of grain length, width and thickness was 2.07%, 0.97%, 1.13%, and the average measurement efficiency was about 9.6 s per grain. In addition, the grain identification model was conducted with 25 grain phenotypic traits, using 6 machine learning methods. The results showed that the best accuracy for filled/unfilled grain classification was 90.184%.The best accuracy for indica and japonica identification was 99.950%, while for different varieties identification was only 47.252%. Therefore, this method was proved to be an efficient and effective way for crop research.

An Intelligent Analysis Method for 3D Wheat Grain and Ventral Sulcus Traits Based on Structured Light Imaging.

The wheat grain three-dimensional (3D) phenotypic characters are of great significance for final yield and variety breeding, and the ventral sulcus traits are the important factors to the wheat flour yield. The wheat grain trait measurements are necessary; however, the traditional measurement method is still manual, which is inefficient, subjective, and labor intensive; moreover, the ventral sulcus traits can only be obtained by destructive measurement. In this paper, an intelligent analysis method based on the structured light imaging has been proposed to extract the 3D wheat grain phenotypes and ventral sulcus traits. First, the 3D point cloud data of wheat grain were obtained by the structured light scanner, and then, the specified point cloud processing algorithms including single grain segmentation and ventral sulcus location have been designed; finally, 28 wheat grain 3D phenotypic characters and 4 ventral sulcus traits have been extracted. To evaluate the best experimental conditions, three-level orthogonal experiments, which include rotation angle, scanning angle, and stage color factors, were carried out on 125 grains of 5 wheat varieties, and the results demonstrated that optimum conditions of rotation angle, scanning angle, and stage color were 30°, 37°, black color individually. Additionally, the results also proved that the mean absolute percentage errors (MAPEs) of wheat grain length, width, thickness, and ventral sulcus depth were 1.83, 1.86, 2.19, and 4.81%. Moreover, the 500 wheat grains of five varieties were used to construct and validate the wheat grain weight model by 32 phenotypic traits, and the cross-validation results showed that the R 2 of the models ranged from 0.77 to 0.83. Finally, the wheat grain phenotype extraction and grain weight prediction were integrated into the specialized software. Therefore, this method was demonstrated to be an efficient and effective way for wheat breeding research.

Two three-dimensional Fe(II) frameworks based on {P4Mo6} tetrameric clusters exhibiting efficient visible-light photocatalytic properties for the degradation of Cr(VI) and methylene blue.

Two three-dimensional frameworks based on the {P4Mo6} unit, H(4,4'-bipy)2[Fe4(PO4)(H2O)4Na6][Fe6(H2O)4][(Mo6O12)(HPO4)3(PO4)(OH)3]4·5H2O (4,4'-bipy = 4,4'-bipyridine) (1) and H3(C12H14N2)4[Fe4(PO4)(H2O)4Na4][Fe2(Mo6O12(HPO4)3(PO4)(OH)3)4]·6H2O (2) were successfully synthesized by varying the solvent. The extended structures of the two compounds were formed by transition metal Fe(II) ions bridging the {P4Mo6}-based tetrameric clusters around [NaXFe4(PO4)] (X = 6 (1), or X = 4 (2)) core. The 4,4'-bipy molecules and in situ generated methyl viologen cations as templates induce the formation of two three-dimensional structures, an 8-connected bcu topology framework for 1 and a 4-connected 2-fold interpenetrating diamond-like topological network for 2, respectively. Additionally, multiform hydrogen bonds are found in the framework and also play an important role in stabilizing the structure. The proton conduction mechanism of the two compounds can be mainly classified as the Grotthuss mechanism; the proton conductivity values are 1.06 × 10-3 S cm-1 for 1 and 3.13 × 10-3 S cm-1 for 2 at 75 °C under 98% relative humidity. The visible-light photocatalytic activity was evaluated by photocatalytic decomposition of Cr(VI) and MB dye, and the removal ratios can reach 95.6% (1) and 82% (2) for Cr(VI), and 98% (1) and 99% (2) for MB.

A SacB-based system for diverse and multiple genome editing in Gluconobacter oxydans.

Gluconobacter oxydans is an important industrial bacterial strain widely used to produce a lot of useful products. However, very few gene editing tools are available for G. oxydans. This study aimed to develop an efficient genome editing method for G. oxydans using SacB as a counter-selectable marker. A plasmid that could express the kanamycin resistance gene in both E. coli and G. oxydans was constructed using the screened shuttle promoter P116. After optimizing the genome editing conditions, the derivative plasmids could be effectively utilized for diverse genome editing, such as gene deletion, insertion, replacement, and in situ modification in G. oxydans WSH-003. In addition, the SacB-based system also achieved multiple gene editing in G. oxydans. Moreover, the genome of the industrial strain G. oxydans WSH-003 was modified and the growth rate and substrate conversion rate of the strain successfully increased using this system. The system could also have potential to be applied in different G. oxydans strains. The process established in this study also provides a reference for constructing genetic tools for many other genetically recalcitrant bacteria.