Metabolic Engineering Communications最新文献

{"title":"PEZy-miner: An artificial intelligence driven approach for the discovery of plastic-degrading enzyme candidates","authors":"","doi":"10.1016/j.mec.2024.e00248","DOIUrl":"10.1016/j.mec.2024.e00248","url":null,"abstract":"<div><p>Plastic waste has caused a global environmental crisis. Biocatalytic depolymerization mediated by enzymes has emerged as an efficient and sustainable alternative for plastic treatment and recycling. However, it is challenging and time-consuming to discover novel plastic-degrading enzymes using conventional cultivation-based or omics methods. There is a growing interest in developing effective computational methods to identify new enzymes with desirable plastic degradation functionalities by exploring the ever-increasing databases of protein sequences. In this study, we designed an innovative machine learning-based framework, named PEZy-Miner, to mine for enzymes with high potential in degrading plastics of interest. Two datasets integrating information from experimentally verified enzymes and homologs with unknown plastic-degrading activity were created respectively, covering eleven types of plastic substrates. Protein language models and binary classification models were developed to predict enzymatic degradation of plastics along with confidence and uncertainty estimation. PEZy-Miner exhibited high prediction accuracy and stability when validated on experimentally verified enzymes. Furthermore, by masking the experimentally verified enzymes and blending them into homolog dataset, PEZy-Miner effectively concentrated the experimentally verified entries by 14∼30 times while shortlisting promising plastic-degrading enzyme candidates. We applied PEZy-Miner to 0.1 million putative sequences, out of which 27 new sequences were identified with high confidence. This study provided a new computational tool for mining and recommending promising new plastic-degrading enzymes.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000178/pdfft?md5=a6ab15db96315a11ed6d106b7d4eb890&pid=1-s2.0-S2214030124000178-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142158312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Production of (R)-citramalate by engineered Saccharomyces cerevisiae","authors":"","doi":"10.1016/j.mec.2024.e00247","DOIUrl":"10.1016/j.mec.2024.e00247","url":null,"abstract":"<div><p>The budding yeast, <em>Saccharomyces cerevisiae</em>, has a high tolerance to organic acids and alcohols, and thus grows well under toxic concentrations of various compounds in the culture medium, potentially allowing for highly efficient compound production. (<em>R</em>)-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. (<em>R</em>)-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike <em>Escherichia coli</em>, <em>S. cerevisiae</em> has organelles, and its intracellular metabolites are compartmentalized, preventing full use of intracellular acetyl-CoA. Therefore, in this study, to increase the amount of cytosolic acetyl-CoA for highly efficient production of (<em>R</em>)-citramalate, we inhibited the transport of cytosolic acetyl-CoA and pyruvate to the mitochondria. We also constructed a heterologous pathway to supply cytosolic acetyl-CoA. Additionally, we attempted to export (<em>R</em>)-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on (<em>R</em>)-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM (<em>R</em>)-citramalate in batch culture flasks. This is the first report of (<em>R</em>)-citramalate production by recombinant <em>S. cerevisiae</em>, and the (<em>R</em>)-citramalate production by recombinant yeast achieved in this study was the highest reported to date.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000166/pdfft?md5=8e77960467f6df90982ae565f50fc7ce&pid=1-s2.0-S2214030124000166-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141985411","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering thioesterase as a driving force for novel itaconate production via its degradation scheme","authors":"","doi":"10.1016/j.mec.2024.e00246","DOIUrl":"10.1016/j.mec.2024.e00246","url":null,"abstract":"<div><p>Incorporation of irreversible steps in pathway design enhances the overall thermodynamic favorability and often leads to better bioconversion yield given functional enzymes. Using this concept, here we constructed the first non-natural itaconate biosynthesis pathway driven by thioester hydrolysis. Itaconate is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Production of itaconate has long relied on the decarboxylation of TCA cycle intermediate cis-aconitate as the only biosynthetic route. Inspired by nature's design of itaconate detoxification, here we engineered a novel itaconate producing pathway orthogonal to native metabolism with no requirement of auxotrophic knock-out. The reversed degradation pathway initiates with pyruvate and acetyl-CoA condensation forming (S)-citramalyl-CoA, followed by its dehydration and isomerization into itaconyl-CoA then hydrolysis into itaconate. Phenylacetyl-CoA thioesterase (PaaI) from <em>Escherichia</em> <em>coli</em> was identified via screening to deliver the highest itaconate formation efficiency when coupled to the reversible activity of citramalate lyase and itaconyl-CoA hydratase. The preference of PaaI towards itaconyl-CoA hydrolysis over acetyl-CoA and (S)-citramalyl-CoA also minimized the inevitable precursor loss due to enzyme promiscuity. With acetate recycling, acetyl-CoA conservation, and condition optimization, we achieved a final itaconate titer of 1 g/L using the thioesterase driven pathway, which is a significant improvement compared to the original degradation pathway based on CoA transferase. This study illustrates the significance of thermodynamic favorability as a design principle in pathway engineering.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000154/pdfft?md5=8638d3ac45e484bb976c59dea9cff40b&pid=1-s2.0-S2214030124000154-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141963669","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study","authors":"Priti Regmi ,&nbsp;Melanie Knesebeck ,&nbsp;Eckhard Boles ,&nbsp;Dirk Weuster-Botz ,&nbsp;Mislav Oreb","doi":"10.1016/j.mec.2024.e00245","DOIUrl":"https://doi.org/10.1016/j.mec.2024.e00245","url":null,"abstract":"<div><p>Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In <em>S. cerevisiae</em>, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L⁻¹ xylitol from 20 g L⁻¹ xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase <em>ALD6</em> gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from <em>Kluyveromyces lactis</em> acted synergistically with <em>ZWF1</em> overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – <em>ZWF1</em> overexpression and deletion of <em>ALD6</em> - also improve the production of <span>l</span>-galactonate from <span>d</span>-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000142/pdfft?md5=7ade0b7c412cf8487310e2ebc5404b91&pid=1-s2.0-S2214030124000142-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141595127","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CFSA: Comparative flux sampling analysis as a guide for strain design","authors":"R.P. van Rosmalen ,&nbsp;S. Moreno-Paz ,&nbsp;Z.E. Duman-Özdamar,&nbsp;M. Suarez-Diez","doi":"10.1016/j.mec.2024.e00244","DOIUrl":"https://doi.org/10.1016/j.mec.2024.e00244","url":null,"abstract":"<div><p>Genome-scale metabolic models of microbial metabolism have extensively been used to guide the design of microbial cell factories, still, many of the available strain design algorithms often fail to produce a reduced list of targets for improved performance that can be implemented and validated in a step-wise manner. We present Comparative Flux Sampling Analysis (CFSA), a strain design method based on the extensive comparison of complete metabolic spaces corresponding to maximal or near-maximal growth and production phenotypes. The comparison is complemented by statistical analysis to identify reactions with altered flux that are suggested as targets for genetic interventions including up-regulations, down-regulations and gene deletions. We applied CFSA to the production of lipids by <em>Cutaneotrichosporon oleaginosus</em> and naringenin by <em>Saccharomyces cerevisiae</em> identifying engineering targets in agreement with previous studies as well as new interventions. CFSA is an easy-to-use, robust method that suggests potential metabolic engineering targets for growth-uncoupled production that can be applied to the design of microbial cell factories.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000130/pdfft?md5=69d0fb5da6998ef5e347063552f98736&pid=1-s2.0-S2214030124000130-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141542319","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression and characterization of monofunctional alcohol dehydrogenase enzymes in Clostridium thermocellum","authors":"Daniela Prates Chiarelli ,&nbsp;Bishal Dev Sharma ,&nbsp;Shuen Hon ,&nbsp;Luana Walravens Bergamo ,&nbsp;Lee R. Lynd ,&nbsp;Daniel G. Olson","doi":"10.1016/j.mec.2024.e00243","DOIUrl":"https://doi.org/10.1016/j.mec.2024.e00243","url":null,"abstract":"<div><p><em>Clostridium thermocellum</em> is a thermophilic anaerobic bacterium that could be used for cellulosic biofuel production due to its strong native ability to consume cellulose, however its ethanol production ability needs to be improved to enable commercial application. In our previous strain engineering work, we observed a spontaneous mutation in the native <em>adhE</em> gene that reduced ethanol production. Here we attempted to complement this mutation by heterologous expression of 18 different alcohol dehydrogenase (<em>adh)</em> genes. We were able to express all of them successfully in <em>C. thermocellum</em>. Surprisingly, however, none of them increased ethanol production, and several actually <em>decreased</em> it. Our findings contribute to understanding the correlation between <em>C. thermocellum</em> ethanol production and Adh enzyme cofactor preferences. The identification of a set of <em>adh</em> genes that can be successfully expressed in this organism provides a foundation for future investigations into how the properties of Adh enzymes affect ethanol production.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000129/pdfft?md5=5d222b62409146f886808888e57c6440&pid=1-s2.0-S2214030124000129-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141480759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"13C-metabolic flux analysis reveals metabolic rewiring in HL-60 neutrophil-like cells through differentiation and immune stimulation","authors":"Takeo Taniguchi ,&nbsp;Nobuyuki Okahashi ,&nbsp;Fumio Matsuda","doi":"10.1016/j.mec.2024.e00239","DOIUrl":"https://doi.org/10.1016/j.mec.2024.e00239","url":null,"abstract":"<div><p>Neutrophils are innate immune cells and the first line of defense for the maintenance of homeostasis. However, our knowledge of the metabolic rewiring associated with their differentiation and immune stimulation is limited. Here, quantitative ¹³C-metabolic flux analysis was performed using HL-60 cells as the neutrophil model. A metabolic model for ¹³C-metabolic flux analysis of neutrophils was developed based on the accumulation of ¹³C in intracellular metabolites derived from ¹³C-labeled extracellular carbon sources and intracellular macromolecules. Aspartate and glutamate in the medium were identified as carbon sources that enter central carbon metabolism. Furthermore, the breakdown of macromolecules, estimated to be fatty acids and nucleic acids, was observed. Based on these results, a modified metabolic model was used for ¹³C-metabolic flux analysis of undifferentiated, differentiated, and lipopolysaccharide (LPS)-activated HL-60 cells. The glucose uptake rate and glycolytic flux decreased with differentiation, whereas the tricarboxylic acid (TCA) cycle flux remained constant. The addition of LPS to differentiated HL-60 cells activated the glucose uptake rate and pentose phosphate pathway (PPP) flux levels, resulting in an increased rate of total NADPH regeneration, which could be used to generate reactive oxygen species. The flux levels of fatty acid degradation and synthesis were also increased in LPS-activated HL-60 cells. Overall, this study highlights the quantitative metabolic alterations in multiple pathways via the differentiation and activation of HL-60 cells using ¹³C-metabolic flux analysis.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000087/pdfft?md5=9e66b20619ea8e938872df783c3173fb&pid=1-s2.0-S2214030124000087-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141243869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering a carbon source-responsive promoter for improved biosynthesis in the non-conventional yeast Kluyveromyces marxianus","authors":"Shane Bassett,&nbsp;Nancy A. Da Silva","doi":"10.1016/j.mec.2024.e00238","DOIUrl":"10.1016/j.mec.2024.e00238","url":null,"abstract":"<div><p>Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast <em>Kluyveromyces marxianus</em>, by fusing carbon-source responsive elements of the native <em>ICL1</em> promoter to the strong <em>S. cerevisiae TDH3</em> or native <em>NC1</em> promoter cores. Two hybrids, P_{<em>IT350</em>} and P_{<em>IN450</em>}, were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the <em>Gerbera hybrida</em> 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of P_{<em>IN450</em>} increased TAL more than 50% relative to the native <em>NC1</em> promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the <em>Penicillium griseofulvum</em> 6-methylsalicylic acid synthase (6-MSAS) under the control of P_{<em>IN450</em>} resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used P_{<em>IN450</em>} to express the <em>Pseudomonas savastanoi</em> IaaM and IaaH proteins and the <em>Salvia pomifera</em> sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for <em>K. marxianus</em>, allowing greater control over heterologous protein expression and improved production of toxic metabolites.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000075/pdfft?md5=e04d392e835f23dae63c13b029270ea3&pid=1-s2.0-S2214030124000075-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141143923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sustainable biosynthesis of squalene from waste cooking oil by the yeast Yarrowia lipolytica","authors":"Shuhui Wang ,&nbsp;Xu Sun ,&nbsp;Yuqing Han ,&nbsp;Zhuo Li ,&nbsp;Xiaocong Lu ,&nbsp;Hongrui Shi ,&nbsp;Cui-ying Zhang ,&nbsp;Adison Wong ,&nbsp;Aiqun Yu","doi":"10.1016/j.mec.2024.e00240","DOIUrl":"https://doi.org/10.1016/j.mec.2024.e00240","url":null,"abstract":"<div><p>Squalene is a highly sought-after triterpene compound in growing demand, and its production offers a promising avenue for circular economy practices. In this study, we applied metabolic engineering principles to enhance squalene production in the nonconventional yeast <em>Yarrowia lipolytica</em>, using waste cooking oil as a substrate. By overexpressing key enzymes in the mevalonate pathway — specifically ERG9 encoding squalene synthase, ERG20 encoding farnesyl diphosphate synthase, and HMGR encoding hydroxy-methyl-glutaryl-CoA reductase — we achieved a yield of 779.9 mg/L of squalene. Further co-overexpression of DGA1, encoding diacylglycerol acyltransferase, and CAT2, encoding carnitine acetyltransferase, in combination with prior metabolic enhancements, boosted squalene production to 1381.4 mg/L in the engineered strain Po1g17. To enhance the supply of the precursor acetyl-CoA and inhibit downstream squalene conversion, we supplemented with 6 g/L pyruvic acid and 0.7 mg/L terbinafine, resulting in an overall squalene titer of 2594.1 mg/L. These advancements underscore the potential for sustainable, large-scale squalene production using <em>Y. lipolytica</em> cell factories, contributing to circular economy initiatives by valorizing waste materials.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000099/pdfft?md5=1108c813c703155bf810e5b6e7073c94&pid=1-s2.0-S2214030124000099-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141286067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic bottlenecks of Pseudomonas taiwanensis VLB120 during growth on d-xylose via the Weimberg pathway","authors":"Philipp Nerke,&nbsp;Jonas Korb,&nbsp;Frederick Haala,&nbsp;Georg Hubmann,&nbsp;Stephan Lütz","doi":"10.1016/j.mec.2024.e00241","DOIUrl":"https://doi.org/10.1016/j.mec.2024.e00241","url":null,"abstract":"<div><p>The microbial production of value-added chemicals from renewable feedstocks is an important step towards a sustainable, bio-based economy. Therefore, microbes need to efficiently utilize lignocellulosic biomass and its dominant constituents, such as <span>d</span>-xylose. <em>Pseudomonas taiwanensis</em> VLB120 assimilates <span>d</span>-xylose via the five-step Weimberg pathway. However, the knowledge about the metabolic constraints of the Weimberg pathway<em>,</em> i.e., its regulation, dynamics, and metabolite fluxes, is limited, which hampers the optimization and implementation of this pathway for bioprocesses. We characterized the Weimberg pathway activity of <em>P. taiwanensis</em> VLB120 in terms of biomass growth and the dynamics of pathway intermediates. In batch cultivations, we found excessive accumulation of the intermediates <span>d</span>-xylonolactone and <span>d</span>-xylonate, indicating bottlenecks in <span>d</span>-xylonolactone hydrolysis and <span>d</span>-xylonate uptake. Moreover, the intermediate accumulation was highly dependent on the concentration of <span>d</span>-xylose and the extracellular pH. To encounter the apparent bottlenecks, we identified and overexpressed two genes coding for putative endogenous xylonolactonases PVLB_05820 and PVLB_12345. Compared to the control strain, the overexpression of PVLB_12345 resulted in an increased growth rate and biomass generation of up to 30 % and 100 %, respectively. Next, <span>d</span>-xylonate accumulation was decreased by overexpressing two newly identified <span>d</span>-xylonate transporter genes, PVLB_18545 and <em>gntP</em> (PVLB_13665). Finally, we combined xylonolactonase overexpression with enhanced uptake of <span>d</span>-xylonate by knocking out the <em>gntP</em> repressor gene <em>gntR</em> (PVLB_13655) and increased the growth rate and biomass yield by 50 % and 24 % in stirred-tank bioreactors, respectively. Our study contributes to the fundamental knowledge of the Weimberg pathway in pseudomonads and demonstrates how to encounter the metabolic bottlenecks of the Weimberg pathway to advance strain developments and cell factory design for bioprocesses on renewable feedstocks.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000105/pdfft?md5=97d4236fe0530871599b5c6105208888&pid=1-s2.0-S2214030124000105-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141314250","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}