Metabolic Engineering Communications最新文献

{"title":"Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli","authors":"Alexander Connor ,&nbsp;Caleb Wigham ,&nbsp;Yang Bai ,&nbsp;Manish Rai ,&nbsp;Sebastian Nassif ,&nbsp;Mattheos Koffas ,&nbsp;R. Helen Zha","doi":"10.1016/j.mec.2023.e00219","DOIUrl":"https://doi.org/10.1016/j.mec.2023.e00219","url":null,"abstract":"<div><p>Spider silk proteins (spidroins) are a remarkable class of biomaterials that exhibit a unique combination of high-value attributes and can be processed into numerous morphologies for targeted applications in diverse fields. Recombinant production of spidroins represents the most promising route towards establishing the industrial production of the material, however, recombinant spider silk production suffers from fundamental difficulties that includes low titers, plasmid instability, and translational inefficiencies. In this work, we sought to gain a deeper understanding of upstream bottlenecks that exist in the field through the production of a panel of systematically varied spidroin sequences in multiple <em>E. coli</em> strains. A restriction on basal expression and specific genetic mutations related to stress responses were identified as primary factors that facilitated higher titers of the recombinant silk constructs. Using these findings, a novel strain of <em>E. coli</em> was created that produces recombinant silk constructs at levels 4–33 times higher than standard BL21(DE3). However, these findings did not extend to a similar recombinant protein, an elastin-like peptide. It was found that the recombinant silk proteins, but not the elastin-like peptide, exert toxicity on the <em>E. coli</em> host system, possibly through their high degree of intrinsic disorder. Along with strain engineering, a bioprocess design that utilizes longer culturing times and attenuated induction was found to raise recombinant silk titers by seven-fold and mitigate toxicity. Targeted alteration to the primary sequence of the recombinant silk constructs was also found to mitigate toxicity. These findings identify multiple points of focus for future work seeking to further optimize the recombinant production of silk proteins and is the first work to identify the intrinsic disorder and subsequent toxicity of certain spidroin constructs as a primary factor related to the difficulties of production.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"16 ","pages":"Article e00219"},"PeriodicalIF":5.2,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50170510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Re-evaluation of the impact of BUD21 deletion on xylose utilization by Saccharomyces cerevisiae","authors":"Venkatachalam Narayanan ,&nbsp;Anders G. Sandström ,&nbsp;Marie F. Gorwa-Grauslund","doi":"10.1016/j.mec.2023.e00218","DOIUrl":"https://doi.org/10.1016/j.mec.2023.e00218","url":null,"abstract":"<div><p>Various rational metabolic engineering and random approaches have been applied to introduce and improve xylose utilization and ethanol productivity by <em>Saccharomyces cerevisiae</em>. Among them, the <em>BUD21</em> gene was identified as an interesting candidate for enhancing xylose consumption as its deletion appeared to be sufficient to improve growth, substrate utilization and ethanol productivity on xylose, even in a laboratory strain lacking a heterologous xylose pathway. The present study aimed at studying the influence of <em>BUD21</em> deletion in recombinant strains carrying heterologous oxido-reductive xylose utilization pathway. The positive effect of <em>BUD21</em> gene deletion on aerobic growth and xylose utilization could not be confirmed in two non-engineered laboratory strains (BY4741 and CEN.PK 113-7D) that were grown in YP rich medium with 20 g/L xylose as sole carbon source, despite the fact that effective deletion of <em>BUD21</em> gene was confirmed using both genotypic (colony PCR) and phenotypic (heat sensitive phenotype of the <em>BUD21</em> deletion mutant) control experiments. Therefore, the effect of <em>BUD21</em> deletion on xylose fermentation might be strain- or medium-dependent.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"16 ","pages":"Article e00218"},"PeriodicalIF":5.2,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50170515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis","authors":"Aurélie Bouin ,&nbsp;Congqiang Zhang ,&nbsp;Nic D. Lindley ,&nbsp;Gilles Truan ,&nbsp;Thomas Lautier","doi":"10.1016/j.mec.2023.e00222","DOIUrl":"10.1016/j.mec.2023.e00222","url":null,"abstract":"<div><p>Fusion of catalytic domains can accelerate cascade reactions by bringing enzymes in close proximity. However, the design of a protein fusion and the choice of a linker are often challenging and lack of guidance. To determine the impact of linker parameters on fusion proteins, a library of linkers featuring various lengths, secondary structures, extensions and hydrophobicities was designed. Linkers were used to fuse the lycopene cyclase (crtY) and β-carotene hydroxylase (crtZ) from <em>Pantoea ananatis</em> to create fusion proteins to produce zeaxanthin. The fusion efficiency was assessed by comparing the carotenoids content in a carotenoid-producer <em>Escherichia coli</em> strain. It was shown that in addition to the orientation of the enzymes and the size of the linker, the first amino acid of the linker is also a key factor in determining the efficiency of a protein fusion. The wide range of sequence diversity in our linker library enables the fine tuning of protein fusion and this approach can be easily transferred to other enzyme couples.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"16 ","pages":"Article e00222"},"PeriodicalIF":5.2,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/bc/34/main.PMC10165439.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9452847","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":"Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS)","authors":"Jasper L.S.P. Driessen ,&nbsp;Josefin Johnsen ,&nbsp;Ivan Pogrebnyakov ,&nbsp;Elsayed T.T. Mohamed ,&nbsp;Solange I. Mussatto ,&nbsp;Adam M. Feist ,&nbsp;Sheila I. Jensen ,&nbsp;Alex T. Nielsen","doi":"10.1016/j.mec.2023.e00223","DOIUrl":"https://doi.org/10.1016/j.mec.2023.e00223","url":null,"abstract":"<div><p>Microbial tolerance to toxic compounds formed during biomass pretreatment is a significant challenge to produce bio-based products from lignocellulose cost effectively. Rational engineering can be problematic due to insufficient prerequisite knowledge of tolerance mechanisms. Therefore, adaptive laboratory evolution was applied to obtain 20 tolerant lineages of <em>Bacillus subtilis</em> strains able to utilize Distiller's Dried Grains with Solubles-derived (DDGS) hydrolysate. Evolved strains showed both improved growth performance and retained heterologous enzyme production using 100% hydrolysate-based medium, whereas growth of the starting strains was essentially absent. Whole-genome resequencing revealed that evolved isolates acquired mutations in the global regulator c<em>odY</em> in 15 of the 19 sequenced isolates. Furthermore, mutations in genes related to oxidative stress (<em>katA</em>, <em>perR</em>) and flagella function appeared in both tolerance and control evolution experiments without toxic compounds. Overall, tolerance adaptive laboratory evolution yielded strains able to utilize DDGS-hydrolysate to produce enzymes and hence proved to be a valuable tool for the valorization of lignocellulose.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"16 ","pages":"Article e00223"},"PeriodicalIF":5.2,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50170514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering sorghum for higher 4-hydroxybenzoic acid content","authors":"Chien-Yuan Lin ,&nbsp;Yang Tian ,&nbsp;Kimberly Nelson-Vasilchik ,&nbsp;Joel Hague ,&nbsp;Ramu Kakumanu ,&nbsp;Mi Yeon Lee ,&nbsp;Venkataramana R. Pidatala ,&nbsp;Jessica Trinh ,&nbsp;Christopher M. De Ben ,&nbsp;Jutta Dalton ,&nbsp;Trent R. Northen ,&nbsp;Edward E.K. Baidoo ,&nbsp;Blake A. Simmons ,&nbsp;John M. Gladden ,&nbsp;Corinne D. Scown ,&nbsp;Daniel H. Putnam ,&nbsp;Albert P. Kausch ,&nbsp;Henrik V. Scheller ,&nbsp;Aymerick Eudes","doi":"10.1016/j.mec.2022.e00207","DOIUrl":"10.1016/j.mec.2022.e00207","url":null,"abstract":"<div><p>Engineering bioenergy crops to accumulate coproducts <em>in planta</em> can increase the value of lignocellulosic biomass and enable a sustainable bioeconomy. In this study, we engineered sorghum with a bacterial gene encoding a chorismate pyruvate-lyase (<em>ubiC</em>) to reroute the plastidial pool of chorismate from the shikimate pathway into the valuable compound 4-hydroxybenzoic acid (4-HBA). A gene encoding a feedback-resistant version of 3-deoxy-<span>d</span>-arabino-heptulonate-7-phosphate synthase (<em>aroG</em>) was also introduced in an attempt to increase the carbon flux through the shikimate pathway. At the full maturity and senesced stage, two independent lines that co-express <em>ubiC</em> and <em>aroG</em> produced 1.5 and 1.7 dw% of 4-HBA in biomass, which represents 36- and 40-fold increases compared to the titer measured in wildtype. The two transgenic lines showed no obvious phenotypes, growth defects, nor alteration of cell wall polysaccharide content when cultivated under controlled conditions. In the field, when harvested before grain maturity, transgenic lines contained 0.8 and 1.2 dw% of 4-HBA, which represent economically relevant titers based on recent technoeconomic analysis. Only a slight reduction (11–15%) in biomass yield was observed in transgenics grown under natural environment. This work provides the first metabolic engineering steps toward 4-HBA overproduction in the bioenergy crop sorghum to improve the economics of biorefineries by accumulating a value-added coproduct that can be recovered from biomass and provide an additional revenue stream.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00207"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9519784/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40393831","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 of Yarrowia lipolytica for terpenoid production","authors":"Jonathan Asmund Arnesen,&nbsp;Irina Borodina","doi":"10.1016/j.mec.2022.e00213","DOIUrl":"10.1016/j.mec.2022.e00213","url":null,"abstract":"<div><p>Terpenoids are a group of chemicals of great importance for human health and prosperity. Terpenoids can be used for human and animal nutrition, treating diseases, enhancing agricultural output, biofuels, fragrances, cosmetics, and flavouring. However, due to the rapid depletion of global natural resources and manufacturing practices relying on unsustainable petrochemical synthesis, there is a need for economic alternatives to supply the world's demand for these essential chemicals. Microbial biosynthesis offers the means to develop scalable and sustainable bioprocesses for terpenoid production. In particular, the non-conventional yeast <em>Yarrowia lipolytica</em> demonstrates excellent potential as a chassis for terpenoid production due to its amenability to industrial production scale-up, genetic engineering, and high accumulation of terpenoid precursors. This review aims to illustrate the scientific progress in developing <em>Y. lipolytica</em> terpenoid cell factories, focusing on metabolic engineering approaches for strain improvement and cultivation optimization.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00213"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9663531/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40691333","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 targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway","authors":"Andrea Steinmann ,&nbsp;Katrin Schullehner ,&nbsp;Anna Kohl ,&nbsp;Christina Dickmeis ,&nbsp;Maurice Finger ,&nbsp;Georg Hubmann ,&nbsp;Guido Jach ,&nbsp;Ulrich Commandeur ,&nbsp;Marco Girhard ,&nbsp;Vlada B. Urlacher ,&nbsp;Stephan Lütz","doi":"10.1016/j.mec.2022.e00205","DOIUrl":"10.1016/j.mec.2022.e00205","url":null,"abstract":"<div><p>Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization.</p><p>In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in <em>E. coli</em>. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant <em>E. coli</em> strains, demonstrating the dynamic accumulation of metabolites.</p><p>The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing <em>E. coli</em>. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00205"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9474286/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40365479","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":"Itaconic acid production is regulated by LaeA in Aspergillus pseudoterreus","authors":"Kyle R. Pomraning ,&nbsp;Ziyu Dai ,&nbsp;Nathalie Munoz ,&nbsp;Young-Mo Kim ,&nbsp;Yuqian Gao ,&nbsp;Shuang Deng ,&nbsp;Teresa Lemmon ,&nbsp;Marie S. Swita ,&nbsp;Jeremy D. Zucker ,&nbsp;Joonhoon Kim ,&nbsp;Stephen J. Mondo ,&nbsp;Ellen Panisko ,&nbsp;Meagan C. Burnet ,&nbsp;Bobbie-Jo M. Webb-Robertson ,&nbsp;Beth Hofstad ,&nbsp;Scott E. Baker ,&nbsp;Kristin E. Burnum-Johnson ,&nbsp;Jon K. Magnuson ,&nbsp;for the Agile BioFoundry","doi":"10.1016/j.mec.2022.e00203","DOIUrl":"10.1016/j.mec.2022.e00203","url":null,"abstract":"<div><p>The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we explored its role in regulation of itaconic acid production in <em>Aspergillus pseudoterreus</em>. To understand its role in regulating metabolism, we deleted and overexpressed <em>laeA,</em> and assessed the transcriptome, proteome, and secreted metabolome prior to and during initiation of phosphate limitation induced itaconic acid production. We found that secondary metabolite clusters, including the itaconic acid biosynthetic gene cluster, are regulated by <em>laeA</em> and that <em>laeA</em> is required for high yield production of itaconic acid. Overexpression of LaeA improves itaconic acid yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that may be present in inexpensive sugar containing feedstocks such as excess phosphate or complex nutrient sources. This suggests that global regulators of metabolism may be useful targets for engineering metabolic flux that is robust to environmental heterogeneity.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00203"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/4e/ad/main.PMC9440423.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40352282","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":"Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides","authors":"Nemailla Bonturi ,&nbsp;Marina Julio Pinheiro ,&nbsp;Paola Monteiro de Oliveira ,&nbsp;Eka Rusadze ,&nbsp;Tobias Eichinger ,&nbsp;Gintare Liudžiūtė ,&nbsp;Juliano Sabedotti De Biaggi ,&nbsp;Age Brauer ,&nbsp;Maido Remm ,&nbsp;Everson Alves Miranda ,&nbsp;Rodrigo Ledesma-Amaro ,&nbsp;Petri-Jaan Lahtvee","doi":"10.1016/j.mec.2022.e00200","DOIUrl":"10.1016/j.mec.2022.e00200","url":null,"abstract":"<div><p><em>Rhodotorula toruloides</em> is a potential chassis for microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast <em>R. toruloides</em>. <em>R. toruloides</em> CCT 0783 was sequenced, and used for the GGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette by using different promoter combinations. The cassettes were transformed into <em>R. toruloides</em> and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for <em>R. toruloides</em>, enabling the efficient design of complex metabolic pathways.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00200"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000098/pdfft?md5=a2fe424d543e5912a0eacd9cd6daa4d6&pid=1-s2.0-S2214030122000098-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46506584","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":"Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production","authors":"Jeffrey J. Czajka ,&nbsp;Deepanwita Banerjee ,&nbsp;Thomas Eng ,&nbsp;Javier Menasalvas ,&nbsp;Chunsheng Yan ,&nbsp;Nathalie Munoz Munoz ,&nbsp;Brenton C. Poirier ,&nbsp;Young-Mo Kim ,&nbsp;Scott E. Baker ,&nbsp;Yinjie J. Tang ,&nbsp;Aindrila Mukhopadhyay","doi":"10.1016/j.mec.2022.e00206","DOIUrl":"10.1016/j.mec.2022.e00206","url":null,"abstract":"<div><p>In this study, a 14-gene edited <em>Pseudomonas putida</em> KT2440 strain for heterologous indigoidine production was examined using three distinct omic datasets. Transcriptomic data indicated that CRISPR/dCpf1-interference (CRISPRi) mediated multiplex repression caused global gene expression changes, implying potential undesirable changes in metabolic flux. ¹³C-metabolic flux analysis (¹³C-MFA) revealed that the core <em>P. putida</em> flux network after CRISPRi repression was conserved, with moderate reduction of TCA cycle and pyruvate shunt activity along with glyoxylate shunt activation during glucose catabolism. Metabolomic results identified a change in intracellular TCA metabolites and extracellular metabolite secretion profiles (sugars and succinate overflow) in the engineered strains. These omic analyses guided further strain engineering, with a random mutagenesis screen first identifying an optimal ribosome binding site (RBS) for Cpf1 that enabled stronger product-substrate pairing (1.6–fold increase). Then, deletion strains were constructed with excision of the PHA operon (Δ<em>phaAZC-IID</em>) resulting in a 2.2–fold increase in indigoidine titer over the optimized Cpf1-RBS construct at the end of the growth phase (∼6 h). The maximum indigoidine titer (at 72 h) in the Δ<em>phaAZC-IID</em> strain had a 1.5–fold and 1.8–fold increase compared to the optimized Cpf1-RBS construct and the original strain, respectively. Overall, this study demonstrated that integration of omic data types is essential for understanding responses to complex metabolic engineering designs and directly quantified the effect of such modifications on central metabolism.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"15 ","pages":"Article e00206"},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/15/32/main.PMC9494242.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33482926","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}