Radiomics-Based Machine Learning Model for Diagnosis of Acute Pancreatitis Using Computed Tomography.

In the early diagnostic workup of acute pancreatitis (AP), the role of contrast-enhanced CT is to establish the diagnosis in uncertain cases, assess severity, and detect potential complications like necrosis, fluid collections, bleeding or portal vein thrombosis. The value of texture analysis/radiomics of medical images has rapidly increased during the past decade, and the main focus has been on oncological imaging and tumor classification. Previous studies assessed the value of radiomics for differentiating between malignancies and inflammatory diseases of the pancreas as well as for prediction of AP severity. The aim of our study was to evaluate an automatic machine learning model for AP detection using radiomics analysis. Patients with abdominal pain and contrast-enhanced CT of the abdomen in an emergency setting were retrospectively included in this single-center study. The pancreas was automatically segmented using TotalSegmentator and radiomics features were extracted using PyRadiomics. We performed unsupervised hierarchical clustering and applied the random-forest based Boruta model to select the most important radiomics features. Important features and lipase levels were included in a logistic regression model with AP as the dependent variable. The model was established in a training cohort using fivefold cross-validation and applied to the test cohort (80/20 split). From a total of 1012 patients, 137 patients with AP and 138 patients without AP were included in the final study cohort. Feature selection confirmed 28 important features (mainly shape and first-order features) for the differentiation between AP and controls. The logistic regression model showed excellent diagnostic accuracy of radiomics features for the detection of AP, with an area under the curve (AUC) of 0.932. Using lipase levels only, an AUC of 0.946 was observed. Using both radiomics features and lipase levels, we showed an excellent AUC of 0.933 for the detection of AP. Automated segmentation of the pancreas and consecutive radiomics analysis almost achieved the high diagnostic accuracy of lipase levels, a well-established predictor of AP, and might be considered an additional diagnostic tool in unclear cases. This study provides scientific evidence that automated image analysis of the pancreas achieves comparable diagnostic accuracy to lipase levels and might therefore be used in the future in the rapidly growing era of AI-based image analysis.

Diagnostic performance of photon-counting detector CT for differentiation between adrenal adenomas and metastases.

OBJECTIVES: Aim of this study was to assess the value of virtual non-contrast (VNC) reconstructions in differentiating between adrenal adenomas and metastases on a photon-counting detector CT (PCD-CT). MATERIAL AND METHODS: Patients with adrenal masses and contrast-enhanced CT scans in portal venous phase were included. Image reconstructions were performed, including conventional VNC (VNCConv) and PureCalcium VNC (VNCPC), as well as virtual monochromatic images (VMI, 40-90 keV) and iodine maps. We analyzed images using semi-automatic segmentation of adrenal lesions and extracted quantitative data. Logistic regression models, non-parametric tests, Bland-Altman plots, and a random forest classifier were used for statistical analyses. RESULTS: The final study cohort consisted of 90 patients (36 female, mean age 67.8 years [range 39-87]) with adrenal lesions (45 adenomas, 45 metastases). Compared to metastases, adrenal adenomas showed significantly lower CT-values in VNCConv and VNCPC (p = 0.007). Mean difference between VNC and true non-contrast (TNC) was 17.67 for VNCConv and 14.85 for VNCPC. Random forest classifier and logistic regression models both identified VNCConv and VNCPC as the best discriminators. When using 26 HU as the threshold in VNCConv reconstructions, adenomas could be discriminated from metastases with a sensitivity of 86.7% and a specificity of 75.6%. CONCLUSION: VNC algorithms overestimate CT values compared to TNC in the assessment of adrenal lesions. However, they allow a reliable discrimination between adrenal adenomas and metastases and could be used in clinical routine in near future with an increased threshold (e.g., 26 HU). Further (multi-center) studies with larger patient cohorts and standardized protocols are required. CLINICAL RELEVANCE STATEMENT: VNC reconstructions overestimate CT values compared to TNC. Using a different threshold (e.g., 26 HU compared to the established 10 HU), VNC has a high diagnostic accuracy for the discrimination between adrenal adenomas and metastases. KEY POINTS: • Virtual non-contrast reconstructions may be promising tools to differentiate adrenal lesions and might save further diagnostic tests. • The conventional and a new calcium-preserving virtual non-contrast algorithm tend to systematically overestimate CT-values compared to true non-contrast images. • Therefore, increasing the established threshold for true non-contrast images (e.g., 10HU) may help to differentiate between adrenal adenomas and metastases on contrast-enhanced CT.

Optimal conspicuity of pancreatic ductal adenocarcinoma in virtual monochromatic imaging reconstructions on a photon-counting detector CT: comparison to conventional MDCT.

PURPOSE: To analyze the conspicuity of pancreatic ductal adenocarcinoma (PDAC) in virtual monoenergetic images (VMI) on a novel photon-counting detector CT (PCD-CT) in comparison to energy-integrating CT (EID-CT). METHODS: Inclusion criteria comprised initial diagnosis of PDAC (reference standard: histopathological analysis) and standardized contrast-enhanced CT imaging either on an EID-CT or a PCD-CT. Patients were excluded due to different histopathological diagnosis or missing tumor delineation on CT. On the PCD-CT, 40-190 keV VMI reconstructions were generated. Image noise, tumor-to-pancreas ratio (TPR) and contrast-to-noise ratio (CNR) were analyzed by ROI-based measurements in arterial and portal venous contrast phase. Two board-certified radiologist evaluated image quality and tumor delineation at both, EID-CT and PCD-CT (40 and 70 keV). RESULTS: Thirty-eight patients (mean age 70.4 years ± 10.3 [range 45-91], 27 males; PCD-CT: n=19, EID-CT: n=19) were retrospectively included. On the PCD-CT, tumor conspicuity (reflected by low TPR and high CNR) was significantly improved at low-energy VMI series (≤ 70 keV compared to > 70 keV), both in arterial and in portal venous contrast phase (P < 0.001), reaching the maximum at 40 keV. Comparison between PCD-CT and EID-CT showed significantly higher CNR on the PCD-CT in portal venous contrast phase at < 70 keV (P < 0.016). On the PCD-CT, tumor conspicuity was improved in portal venous contrast phase compared to arterial contrast phase especially at the lower end of the VMI spectrum (≤ 70 keV). Qualitative analysis revealed that tumor delineation is improved in 40 keV reconstructions compared to 70 keV reconstructions on a PCD-CT. CONCLUSION: PCD-CT VMI reconstructions (≤ 70 keV) showed significantly improved conspicuity of PDAC in quantitative and qualitative analysis in both, arterial and portal venous contrast phase, compared to EID-CT, which may be important for early detection of tumor tissue in clinical routine. Tumor delineation was superior in portal venous contrast phase compared to arterial contrast phase.

Image Characteristics of Virtual Non-Contrast Series Derived from Photon-Counting Detector Coronary CT Angiography-Prerequisites for and Feasibility of Calcium Quantification.

In photon-counting detector CT (PCD-CT), coronary artery calcium scoring (CACS) can be performed using virtual non-contrast (VNC) series derived from coronary CT angiography (CCTA) datasets. Our study analyzed image characteristics of VNC series in terms of the efficacy of virtual iodine "removal" and image noise to determine whether the prerequisites for calcium quantification were satisfied. We analyzed 38 patients who had undergone non-enhanced CT followed by CCTA on a PCD-CT. VNC reconstructions were performed at different settings and algorithms (conventional VNCConv; PureCalcium VNCPC). Virtual iodine "removal" was investigated by comparing histograms of heart volumes. Noise was assessed within the left ventricular cavity. Calcium was quantified on the true non-contrast (TNC) and all VNC series. The histograms were comparable for TNC and all VNC. Image noise between TNC and all VNC differed slightly but significantly. VNCConv CACS showed a significant underestimation regardless of the reconstruction setting, while VNCPC CACS were comparable to TNC. Correlations between TNC and VNC were excellent, with a higher predictive accuracy for VNCPC. In conclusion, the iodine contrast can be effectively subtracted from CCTA datasets. The remaining VNC series satisfy the requirements for CACS, yielding results with excellent correlation compared to TNC-based CACS and high predicting accuracy.

Anemia detection and quantification in contrast-enhanced CT scans on a novel photon-counting detector CT: A prospective proof-of-concept study.

PURPOSE: To correlate CT values of the blood pool on VNC series with serum hemoglobin values for the detection of anemia in oncologic patients undergoing contrast-enhanced PCD-CT scans. METHODS: This prospective study (NCT04989192) included consecutive oncologic patients undergoing contrast-enhanced CT on a novel PCD-CT system between 08/2021 and 01/2022. The interval between complete blood count (CBC) and CT scan acquisition had to be no more than seven days. CT-values of the blood pool were measured on 70 keV VMI series (CT-values(BP)70keV) and on VNC series (CT-values(BP)VNC) at five anatomic positions (left atrium, left ventricle, main pulmonary artery, ascending and descending aorta) and averaged per patient. Pearson correlation analyses and ROC analyses were performed to identify relations between CT-values(BP)VNC, CBC parameters, and degrees of anemia as defined by the WHO (no anemia, mild, moderate, severe anemia). RESULTS: A total of 329 patients (age 68 ± 12 years; 200 men) were included. CT-values(BP)VNC showed a strong linear correlation to serum hemoglobin (r2 = 0.80, p <.001) and hematocrit (r2 = 0.76, p <.001) and were significantly different between anemia subgroups in both women and men (ΔHU: 3.5-11.4; all p <.01). ROC analyses yielded high diagnostic performance for the identification of patients without anemia, patients without and with mild anemia, and patients with severe anemia using gender-specific cutoffs for CT-value(BP)VNC (all AUC's > 0.90). CONCLUSIONS: The spectral information inherent in PCD-CT acquisitions allows the detection and quantification of anemia in contrast-enhanced CT acquisitions of oncologic patients with high diagnostic accuracy.

Systems metabolic engineering upgrades Corynebacterium glutamicum for selective high-level production of the chiral drug precursor and cell-protective extremolyte L-pipecolic acid.

The nonproteinogenic cyclic metabolite l-pipecolic acid is a chiral precursor for the synthesis of various commercial drugs and functions as a cell-protective extremolyte and mediator of defense in plants, enabling high-value applications in the pharmaceutical, medical, cosmetic, and agrochemical markets. To date, the production of the compound is unfavorably fossil-based. Here, we upgraded the strain Corynebacterium glutamicum for l-pipecolic acid production using systems metabolic engineering. Heterologous expression of the l-lysine 6-dehydrogenase pathway, apparently the best route to be used in the microbe, yielded a family of strains that enabled successful de novo synthesis from glucose but approached a limit of performance at a yield of 180 mmol mol-1. Detailed analysis of the producers at the transcriptome, proteome, and metabolome levels revealed that the requirements of the introduced route were largely incompatible with the cellular environment, which could not be overcome after several further rounds of metabolic engineering. Based on the gained knowledge, we based the strain design on l-lysine 6-aminotransferase instead, which enabled a substantially higher in vivo flux toward l-pipecolic acid. The tailormade producer C. glutamicum PIA-7 formed l-pipecolic acid up to a yield of 562 mmol mol-1, representing 75% of the theoretical maximum. Ultimately, the advanced mutant PIA-10B achieved a titer of 93 g L-1 in a fed-batch process on glucose, outperforming all previous efforts to synthesize this valuable molecule de novo and even approaching the level of biotransformation from l-lysine. Notably, the use of C. glutamicum allows the safe production of GRAS-designated l-pipecolic acid, providing extra benefit toward addressing the high-value pharmaceutical, medical, and cosmetic markets. In summary, our development sets a milestone toward the commercialization of biobased l-pipecolic acid.

High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions.

BACKGROUND: Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. RESULTS: Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L-1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. CONCLUSIONS: The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production.

Systems metabolic engineering upgrades Corynebacterium glutamicum to high-efficiency cis, cis-muconic acid production from lignin-based aromatics.

Lignin displays a highly challenging renewable. To date, massive amounts of lignin, generated in lignocellulosic processing facilities, are for the most part merely burned due to lacking value-added alternatives. Aromatic lignin monomers of recognized relevance are in particular vanillin, and to a lesser extent vanillate, because they are accessible at high yield from softwood-lignin using industrially operated alkaline oxidative depolymerization. Here, we metabolically engineered C. glutamicum towards cis, cis-muconate (MA) production from these key aromatics. Starting from the previously created catechol-based producer C. glutamicum MA-2, systems metabolic engineering first discovered an unspecific aromatic aldehyde reductase that formed aromatic alcohols from vanillin, protocatechualdehyde, and p- hydroxybenzaldehyde, and was responsible for the conversion up to 57% of vanillin into vanillyl alcohol. The alcohol was not re-consumed by the microbe later, posing a strong drawback on the producer. The identification and subsequent elimination of the encoding fudC gene completely abolished vanillyl alcohol formation. Second, the initially weak flux through the native vanillin and vanillate metabolism was enhanced up to 2.9-fold by implementing synthetic pathway modules. Third, the most efficient protocatechuate decarboxylase AroY for conversion of the midstream pathway intermediate protocatechuate into catechol was identified out of several variants in native and codon optimized form and expressed together with the respective helper proteins. Fourth, the streamlined modules were all genomically combined which yielded the final strain MA-9. MA-9 produced bio-based MA from vanillin, vanillate, and seven structurally related aromatics at maximum selectivity. In addition, MA production from softwood-based vanillin, obtained through alkaline depolymerization, was demonstrated.

Assessment of epicardial adipose tissue on virtual non-contrast images derived from photon-counting detector coronary CTA datasets.

OBJECTIVES: To assess epicardial adipose tissue (EAT) volume and attenuation of different virtual non-contrast (VNC) reconstructions derived from coronary CTA (CCTA) datasets of a photon-counting detector (PCD) CT-system to replace true non-contrast (TNC) series. METHODS: Consecutive patients (n = 42) with clinically indicated CCTA and coronary TNC were included. Two VNC series were reconstructed, using a conventional (VNCConv) and a novel calcium-preserving (VNCPC) algorithm. EAT was segmented on TNC, VNCConv, VNCPC, and CCTA (CTA-30) series using thresholds of -190 to -30 HU and an additional segmentation on the CCTA series with an upper threshold of 0 HU (CTA0). EAT volumes and their histograms were assessed for each series. Linear regression was used to correlate EAT volumes and the Euclidian distance for histograms. The paired t-test and the Wilcoxon signed-rank test were used to assess differences for parametric and non-parametric data. RESULTS: EAT volumes from VNC and CCTA series showed significant differences compared to TNC (all p < .05), but excellent correlation (all R2 > 0.9). Measurements on the novel VNCPC series showed the best correlation (R2 = 0.99) and only minor absolute differences compared to TNC values. Mean volume differences were -12%, -3%, -13%, and +10% for VNCConv, VNCPC, CTA-30, and CTA0 compared to TNC. Distribution of CT values on VNCPC showed less difference to TNC than on VNCConv (mean attenuation difference +7% vs. +2%; Euclidean distance of histograms 0.029 vs. 0.016). CONCLUSIONS: VNCPC-reconstructions of PCD-CCTA datasets can be used to reliably assess EAT volume with a high accuracy and only minor differences in CT values compared to TNC. Substitution of TNC would significantly decrease patient's radiation dose. KEY POINTS: • Measurement of epicardial adipose tissue (EAT) volume and attenuation are feasible on virtual non-contrast (VNC) series with excellent correlation to true non-contrast series (all R2>0.9). • Differences in VNC algorithms have a significant impact on EAT volume and CT attenuation values. • A novel VNC algorithm (VNCPC) enables reliable assessment of EAT volume and attenuation with superior accuracy compared to measurements on conventional VNC- and CCTA-series.

High-efficiency production of 5-hydroxyectoine using metabolically engineered Corynebacterium glutamicum.

BACKGROUND: Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing. RESULTS: Here, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectDPST achieved a titre of 74 g L-1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABCopt, was successfully achieved without intermediate purification. CONCLUSIONS: C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar.

Systems metabolic engineering of Corynebacterium glutamicum eliminates all by-products for selective and high-yield production of the platform chemical 5-aminovalerate.

5-aminovalerate (AVA) is a platform chemical of substantial commercial value to derive nylon-5 and five-carbon derivatives like δ-valerolactam, 1,5-pentanediol, glutarate, and 5-hydroxyvalerate. Denovo bio-production synthesis of AVA using metabolically engineered cell factories is regarded as exemplary route to provide this chemical in a sustainable way. So far, this route is limited by low titers, rates and yields and suffers from high levels of by-products. To overcome these limitations, we developed a novel family of AVA producing C. glutamicum cell factories. Stepwise optimization included (i) improved AVA biosynthesis by expression balancing of the heterologous davBA genes from P. putida, (ii) reduced formation of the by-product glutarate by disruption of the catabolic y-aminobutyrate pathway (iii), increased AVA export, and (iv) reduced AVA re-import via native and heterologous transporters to account for the accumulation of intracellular AVA up to 300 mM. Strain C. glutamicum AVA-5A, obtained after several optimization rounds, produced 48.3 g L-1 AVA in a fed-batch process and achieved a high yield of 0.21 g g-1. Surprisingly in later stages, the mutant suddenly accumulated glutarate to an extent equivalent to 30% of the amount of AVA formed, tenfold more than in the early process, displaying a severe drawback toward industrial production. Further exploration led to the discovery that ArgD, naturally aminating N-acetyl-l-ornithine during l-arginine biosynthesis, exhibits deaminating side activity on AVA towards glutarate formation. This promiscuity became relevant because of the high intracellular AVA level and the fact that ArgD became unoccupied with the gradually stronger switch-off of anabolism during production. Glutarate formation was favorably abolished in the advanced strains AVA-6A, AVA-6B, and AVA-7, all lacking argD. In a fed-batch process, C. glutamicum AVA-7 produced 46.5 g L-1 AVA at a yield of 0.34 g g-1 and a maximum productivity of 1.52 g L-1 h-1, outperforming all previously reported efforts and stetting a milestone toward industrial manufacturing of AVA. Notably, the novel cell factories are fully genome-based, offering high genetic stability and requiring no selection markers.

Anemia Detection by Hemoglobin Quantification on Contrast-enhanced Photon-counting CT Data Sets.

See also the editorial by Dodd and MacDermott in this issue.

Artificial Intelligence-Based Detection of Pneumonia in Chest Radiographs.

Artificial intelligence is gaining increasing relevance in the field of radiology. This study retrospectively evaluates how a commercially available deep learning algorithm can detect pneumonia in chest radiographs (CR) in emergency departments. The chest radiographs of 948 patients with dyspnea between 3 February and 8 May 2020, as well as 15 October and 15 December 2020, were used. A deep learning algorithm was used to identify opacifications associated with pneumonia, and the performance was evaluated by using ROC analysis, sensitivity, specificity, PPV and NPV. Two radiologists assessed all enrolled images for pulmonal infection patterns as the reference standard. If consolidations or opacifications were present, the radiologists classified the pulmonal findings regarding a possible COVID-19 infection because of the ongoing pandemic. The AUROC value of the deep learning algorithm reached 0.923 when detecting pneumonia in chest radiographs with a sensitivity of 95.4%, specificity of 66.0%, PPV of 80.2% and NPV of 90.8%. The detection of COVID-19 pneumonia in CR by radiologists was achieved with a sensitivity of 50.6% and a specificity of 73%. The deep learning algorithm proved to be an excellent tool for detecting pneumonia in chest radiographs. Thus, the assessment of suspicious chest radiographs can be purposefully supported, shortening the turnaround time for reporting relevant findings and aiding early triage.

Optimal Conspicuity of Liver Metastases in Virtual Monochromatic Imaging Reconstructions on a Novel Photon-Counting Detector CT-Effect of keV Settings and BMI.

In dual-energy CT datasets, the conspicuity of liver metastases can be enhanced by virtual monoenergetic imaging (VMI) reconstructions at low keV levels. Our study investigated whether this effect can be reproduced in photon-counting detector CT (PCD-CT) datasets. We analyzed 100 patients with liver metastases who had undergone contrast-enhanced CT of the abdomen on a PCD-CT (n = 50) or energy-integrating detector CT (EID-CT, single-energy mode, n = 50). PCD-VMI-reconstructions were performed at various keV levels. Identical regions of interest were positioned in metastases, normal liver, and other defined locations assessing image noise, tumor-to-liver ratio (TLR), and contrast-to-noise ratio (CNR). Patients were compared inter-individually. Subgroup analyses were performed according to BMI. On the PCD-CT, noise and CNR peaked at the low end of the keV spectrum. In comparison with the EID-CT, PCD-VMI-reconstructions exhibited lower image noise (at 70 keV) but higher CNR (for ≤70 keV), despite similar CTDIs. Comparing high- and low-BMI patients, CTDI-upregulation was more modest for the PCD-CT but still resulted in similar noise levels and preserved CNR, unlike the EID-CT. In conclusion, PCD-CT VMIs in oncologic patients demonstrated reduced image noise-compared to a standard EID-CT-and improved conspicuity of hypovascularized liver metastases at low keV values. Patients with higher BMIs especially benefited from constant image noise and preservation of lesion conspicuity, despite a more moderate upregulation of CTDI.

GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways.

BACKGROUND: Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. RESULTS: Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. CONCLUSIONS: The developed approach, based on parallel 13C tracer studies with GC-MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum.

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering.

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L-1l-lysine from mannitol at a yield of 0.26 mol mol-1 and a maximum productivity of 2.1 g L-1 h-1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol-1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol-1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being.

The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity.

Microbial production of polyunsaturated fatty acids - high-value ingredients for aquafeed, superfoods, and pharmaceuticals.

Polyunsaturated fatty acids (PUFAs), primarily docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have received worldwide attention in recent years due to an increasing awareness of their uniqueness in improving diet and human health and their apparently inevitable shortage in global availability. Microbial cell factories are a major solution to supplying these precious molecules in sufficient amounts and providing PUFA-rich aquafeed, superfoods, and medical formulations. This review assesses the PUFA world markets and highlights recent advances in upgrading and streamlining microalgae, yeasts, fungi, and bacteria for high-level PUFA production and broadening of the PUFA spectrum.

Channelling carbon flux through the meta-cleavage route for improved poly(3-hydroxyalkanoate) production from benzoate and lignin-based aromatics in Pseudomonas putida H.

Lignin-based aromatics are attractive raw materials to derive medium-chain length poly(3-hydroxyalkanoates) (mcl-PHAs), biodegradable polymers of commercial value. So far, this conversion has exclusively used the ortho-cleavage route of Pseudomonas putida KT2440, which results in the secretion of toxic intermediates and limited performance. Pseudomonas putida H exhibits the ortho- and the meta-cleavage pathways where the latter appears promising because it stoichiometrically yields higher levels of acetyl-CoA. Here, we created a double-mutant H-ΔcatAΔA2 that utilizes the meta route exclusively and synthesized 30% more PHA on benzoate than the parental strain but suffered from catechol accumulation. The single deletion of the catA2 gene in the H strain provoked a slight attenuation on the enzymatic capacity of the ortho route (25%) and activation of the meta route by nearly 8-fold, producing twice as much mcl-PHAs compared to the wild type. Inline, the mutant H-ΔcatA2 showed a 2-fold increase in the intracellular malonyl-CoA abundance - the main precursor for mcl-PHAs synthesis. As inferred from flux simulation and enzyme activity assays, the superior performance of H-ΔcatA2 benefited from reduced flux through the TCA cycle and malic enzyme and diminished by-product formation. In a benzoate-based fed-batch, P. putida H-ΔcatA2 achieved a PHA titre of 6.1 g l-1 and a volumetric productivity of 1.8 g l-1 day-1 . Using Kraft lignin hydrolysate as feedstock, the engineered strain formed 1.4 g l- 1 PHA. The balancing of carbon flux between the parallel catechol-degrading routes emerges as an important strategy to prevent intermediate accumulation and elevate mcl-PHA production in P. putida H and, as shown here, sets the next level to derive this sustainable biopolymer from lignin hydrolysates and aromatics.