Biotechnology and Bioengineering最新文献

{"title":"Manufacturing Process Intensification of Adeno-Associated Viral Vectors Type-8 Using Weak Partitioning Chromatography With UV-Based Process Analytical Technology (PAT)","authors":"Garima Thakur,&nbsp;Sheldon Mink,&nbsp;Hanne Bak,&nbsp;Andrew D. Tustian","doi":"10.1002/bit.70021","DOIUrl":"10.1002/bit.70021","url":null,"abstract":"<div>\u0000 \u0000 <p>Separation of full and empty capsids is a critical step in manufacturing of recombinant adeno-associated viral vector (AAV) based gene therapies. Anion exchange chromatography (AEX) is well-established as a scalable method for full/empty separations. Due to the small differences in surface charge between full and empty capsids, shallow linear gradients of increasing conductivity are commonly used to resolve the two species. However, the resolution is adversely impacted by higher column loading, and most linear gradient processes load at 2e13–2e14 capsids/mL of monolith volume (cp/mL monolith) to achieve full capsid purity targets. With the rapid increase reported in upstream AAV titers up to &gt; 1e12 vg/mL in the bioreactor over the last few years, current downstream purification processes utilizing linear gradient AEX are rapidly approaching scalability limits. This is particularly true for processes utilizing monoliths and membranes which are preferred over resins in AAV purifications due to elimination of pore size diffusion limitations. There is a pressing need for next-generation processes that can load more material in the range of &gt; 1e15 cp/mL, while maintaining operating time and process robustness suitable for GMP manufacturing. This study presents a scalable method for empty/full separations for AAV8 using weak partitioning AEX combined with isocratic elution, demonstrated on CIM QA monoliths which are currently extensively used in the industry for AAV manufacturing. By optimizing load conditions and automating the process with UV-based signals, the strategy achieves &gt; 80% full capsid purity and &gt; 80% genomic yields. Compared to standard linear gradient AEX, the weak partitioning method achieves higher % full capsids as well as higher genomic yield due to combined effects of (i) removal of empty capsids in the flowthrough, (ii) reduction of nonspecific binding interactions as a result of column overloading, and (iii) elimination of peak-cutting by using isocratic elution. The approach enables &gt; 10-fold higher loading per cycle while reducing processing time by 10-fold and is well-suited to rapid cycling or continuous processing operations to support next-generation, high-titer processes.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 10","pages":"2832-2849"},"PeriodicalIF":3.6,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144652963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Scaling-up the Bioconversion of Lignin to 2,4-Pyridinedicarboxylic Acid With Engineered Pseudomonas putida for Bio-Based Plastics Production","authors":"Jan Seeger,&nbsp;Susanne Müller,&nbsp;Helena Gómez-Álvarez,&nbsp;Goran M. M. Rashid,&nbsp;Timothy D. H. Bugg,&nbsp;Eduardo Díaz,&nbsp;Ralf Takors","doi":"10.1002/bit.70020","DOIUrl":"10.1002/bit.70020","url":null,"abstract":"<p>2,4-pyridinedicarboxylic acid (PDCA) is a promising bio-based compound to substitute petroleum-derived terephthalic acid in plastics. It is produced through the microbial conversion of lignin substrates with engineered microorganisms like <i>Pseudomonas putida</i>. To this point, an efficient bioproduction process for PDCA has not yet been established. In this study, we optimized PDCA production with engineered <i>P. putida ligAB</i> and demonstrated bioproduction at up to 30 L scale. PDCA was produced with a volumetric productivity of 390 mg/L/h from the precursor protocatechuate and the product titer was doubled compared to previously reported work. Lignin feedstock and pretreatment combinations were screened to access lignin as substrate for PDCA production. Sodium hydroxide lignin with alkali + heat pretreatment yielded 33 mg/L PDCA at a production rate of 0.1 mg/g/h. Low PDCA production rates could be overcome by developing a bacterial mixed culture by adding the engineered strain <i>Rhodococcus jostii ΔpcaHG</i> that supplies PCA from lignin degradation. The mixed culture increased PDCA productivity of <i>P. putida ligAB</i> by factor 19 (1.9 mg/g_{<i>P. putida</i>}/h).</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 10","pages":"2770-2780"},"PeriodicalIF":3.6,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144622023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Bioprocess Engineering Approach to Boost Selection of Fully Segregated Transformants in Cyanobacteria","authors":"Cecilia Salvagnini,&nbsp;Eliana Gasparotto,&nbsp;Veronica Lucato,&nbsp;Elisabetta Bergantino,&nbsp;Matteo Ballottari,&nbsp;Elena Barbera,&nbsp;Nico Betterle,&nbsp;Eleonora Sforza","doi":"10.1002/bit.70024","DOIUrl":"10.1002/bit.70024","url":null,"abstract":"<p>Cyanobacteria are photoautotrophic microorganisms with significant applications in biotechnology. Although many cyanobacteria, including <i>Picosynechococcus</i> sp. (formerly called <i>Synechococcus</i> sp.) PCC 11901 (<i>Picosynechococcus</i>) and <i>Synechocystis</i> sp. PCC 6803 (<i>Synechocystis</i>), are readily and naturally transformable, their polyploidy poses a major challenge. To obtain a stable phenotype, transgenic strains must be fully segregated, i.e. mutations must appear in all chromosome copies. Traditional protocols rely on re-streaking of colonies on increasingly selective plates, a time-intensive laboratory procedure that requires continuous intervention from the operator. This study proposes an alternative protocol that combines transformation in a batch system in liquid culture with transformant selection in a continuous-flow stirred-tank reactor system. This protocol led to the successful selection of homoplasmic transformants of <i>Picosynechococcus</i> containing, alternatively, an antibiotic resistance alone (construct “SmR”) or a more complex construct (“bKT”) that leads to the accumulation of a ketocarotenoid. The stability of SmR transformants under semi-continuous cultivation in the absence of antibioticsf was tested for 42 days, proving their potential fitness to industrial cultivation conditions. The selection process was also validated on the model species <i>Synechocystis</i>, demonstrating its applicability to other cyanobacterial strains.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 10","pages":"2781-2790"},"PeriodicalIF":3.6,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70024","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144622025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Regulating Transport Preferences of Fucosylated Sugars: Revealing Transport Mechanisms via Sugar Efflux Transporter A Transformation Into a “Tight-in, Tight-out” Mode","authors":"Liu Wenxian,&nbsp;Sun Shengjie,&nbsp;Peng Jing,&nbsp;Zou Sini,&nbsp;Cheng Haina,&nbsp;Chen Zhu,&nbsp;Wang Yuguang,&nbsp;Zhou Hongbo","doi":"10.1002/bit.70019","DOIUrl":"10.1002/bit.70019","url":null,"abstract":"<div>\u0000 \u0000 <p>Sugar transporters play a crucial role in cellular metabolism across diverse organisms, regulating essential biological processes through efficient substrate transport. Despite extensive research efforts, the structures and mechanisms of transporters responsible for sugars have remained elusive. In this study, we investigated the transport efficiency of the <i>Escherichia coli</i> sugar efflux transporter A (SetA) for lactose and fucosylated lactose. By employing site and combinatorial mutations, we obtained a mutant exhibiting approximately sixfold enhanced transporter efficiency for fucosylated lactose while retaining its potency for lactose transport. In this mutant, the fundamental amino acids responsible for recognizing the galactosyl moiety remained unchanged, yet the introduction of two face-to-face aromatic ring residues facilitated the enhanced recognition of the fucosyl moiety. This indicated the transformation of SetA from a universal transporter into a specific “tight-in, tight-out” transporter. Utilizing SetA-based structural modeling, we mapped and investigated mutations associated with diseases. The structural and biochemical insights from SET in this study offer a valuable investigating framework for understanding substrate specificity mechanisms of fucosylated sugar transporters and, by extension, other transporters in broader contexts.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 10","pages":"2675-2687"},"PeriodicalIF":3.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144603105","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Setup for Automatic Raman Measurements in High-Throughput Experimentation","authors":"Christoph Lange,&nbsp;Simon Seidel,&nbsp;Madeline Altmann,&nbsp;Daniel Stors,&nbsp;Annina Kemmer,&nbsp;Linda Cai,&nbsp;Stefan Born,&nbsp;Peter Neubauer,&nbsp;M. Nicolas Cruz Bournazou","doi":"10.1002/bit.70006","DOIUrl":"10.1002/bit.70006","url":null,"abstract":"&lt;p&gt;High-throughput (HT) experimentation is transforming biotechnology by enabling systematic exploration of complex multi-dimensional experimental conditions. However, current analytical methods are often unable to handle the rapid pace of sample generation in HT workflows. This study presents an integrated system of physical devices and software to automate and accelerate Raman spectral measurements in HT-facilities. The setup simultaneously handles eight parallel &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 \u0000 &lt;mrow&gt;\u0000 &lt;mn&gt;50&lt;/mn&gt;\u0000 &lt;mspace&gt;&lt;/mspace&gt;\u0000 \u0000 &lt;mi&gt;μ&lt;/mi&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt;L samples delivered by a pipetting robot, completing measurement, handling, cleaning, and concentration prediction within 45 s per sample. We introduce a machine learning model to predict metabolite concentrations from Raman spectra, achieving mean absolute errors of &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 \u0000 &lt;mrow&gt;\u0000 &lt;mn&gt;0.27&lt;/mn&gt;\u0000 &lt;mspace&gt;&lt;/mspace&gt;\u0000 \u0000 &lt;msup&gt;\u0000 &lt;mstyle&gt;\u0000 &lt;mspace&gt;&lt;/mspace&gt;\u0000 \u0000 &lt;mtext&gt;g L&lt;/mtext&gt;\u0000 &lt;mspace&gt;&lt;/mspace&gt;\u0000 &lt;/mstyle&gt;\u0000 \u0000 &lt;mrow&gt;\u0000 &lt;mo&gt;−&lt;/mo&gt;\u0000 \u0000 &lt;mn&gt;1&lt;/mn&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/msup&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt; for glucose and &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 \u0000 &lt;mrow&gt;\u0000 &lt;mn&gt;0.06&lt;/mn&gt;\u0000 &lt;mspace&gt;&lt;/mspace&gt;\u0000 \u0000 &lt;msup&gt;\u0000 &lt;mstyle&gt;\u0000 &lt;mspace&gt;&lt;/mspace&gt;\u0000 \u0000 &lt;mtext&gt;g L&lt;/mtext&gt;\u0000 &lt;mspace&gt;&lt;/mspace&gt;\u0000 &lt;/mstyle&gt;\u0000 \u0000 &lt;mrow&gt;\u0000 &lt;mo&gt;−&lt;/mo&gt;\u0000 \u0000 &lt;mn&gt;1&lt;/mn&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/msup&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt; for acetate during &lt;i&gt;Escherichia coli&lt;/i&gt; cultivations. This approach enables consistent high-throughput spectral data collection for fermentation monitoring, calibration, and offline analysis, supporting the generatio","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 10","pages":"2751-2769"},"PeriodicalIF":3.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144603104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineered Coenzyme A Biosynthesis and Butyrate Transporter Drives High-Efficient Butyrate Synthesis in Escherichia coli","authors":"Jinhui Li,&nbsp;Yi Xing,&nbsp;Xin Wang,&nbsp;Tong Zhu,&nbsp;Feiyu Fan,&nbsp;Hongtao Xu,&nbsp;Peipei Han,&nbsp;Jun Cai,&nbsp;Xinna Zhu,&nbsp;Xueli Zhang","doi":"10.1002/bit.70018","DOIUrl":"10.1002/bit.70018","url":null,"abstract":"<div>\u0000 \u0000 <p>Butyric acid is a four-carbon fatty acid with a range of applications in chemical, food and pharmaceutical industries. In this study, a heterologous butyrate biosynthetic pathway was engineered in <i>Escherichia coli</i>, which was afflicted by low titer and low yield. To address these issues, two key strategies for metabolic engineering of <i>E. coli</i> were implemented by enhancing coenzyme A (CoA) biosynthesis and optimizing butyrate transport. First, the CoA biosynthesis pathway was engineered through alleviating CoA-mediated inhibition, and enhancing the supply of pantothenate and cysteine precursors. Second, a TolC-associated MdtEF efflux pumps was identified and optimized to mitigate butyrate reuptake. The combined implementation in strain JH016 led to 11.1-fold and 86% increase of butyrate titer and yield, resulting in production of 21.12 g/L butyrate with a yield of 0.95 mol/mol. Our results suggested that CoA engineering and butyrate transporter optimization had a synergistic effect on butyrate production. Furthermore, these strategies could be broadly utilized for the production of various other useful chemicals in the fields of metabolic engineering and synthetic biology.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 10","pages":"2850-2861"},"PeriodicalIF":3.6,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144603349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inside Front Cover Image, Volume 122, Number 8, August 2025","authors":"Elnaz Khorasani,&nbsp;Bahman Vahidi","doi":"10.1002/bit.70023","DOIUrl":"10.1002/bit.70023","url":null,"abstract":"<p>The cover image is based on the article <i>3D-printed scaffolds for cranial bone regeneration: A systematic review of design, materials, and computational optimization</i> by Elnaz Khorasani et al., https://doi.org/10.1002/bit.28994.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70023","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144573741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biotechnology and Bioengineering: Volume 122, Number 8, August 2025","authors":"","doi":"10.1002/bit.28748","DOIUrl":"10.1002/bit.28748","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":"1943-1946"},"PeriodicalIF":3.6,"publicationDate":"2025-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28748","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144573742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Front Cover Image, Volume 122, Number 8, August 2025","authors":"Anton Lindig,&nbsp;Georg Hubmann,&nbsp;Stephan Lütz","doi":"10.1002/bit.70022","DOIUrl":"10.1002/bit.70022","url":null,"abstract":"<p>The cover image is based on the article <i>Microtiter plate cultivation systems enable chemically diverse metabolic footprints during bacterial natural product discovery</i> by Anton Lindig et al., https://doi.org/10.1002/bit.29002.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70022","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144573740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sulfate Reduction in the Hydrogen-Based Membrane Biofilm Reactor Receiving Calcium Reduced Phosphogypsum Water","authors":"Anwar Alsanea,&nbsp;Ayoub Bounaga,&nbsp;Karim Lyamlouli,&nbsp;Youssef Zeroual,&nbsp;Rachid Boulif,&nbsp;Chen Zhou,&nbsp;Bruce Rittmann","doi":"10.1002/bit.70015","DOIUrl":"10.1002/bit.70015","url":null,"abstract":"<div>\u0000 \u0000 <p>Phosphogypsum (PG), a byproduct of phosphate mining, contains sulfate that can be leached and converted to elemental sulfur, thus offering a sustainable opportunity to recover sulfur (S) as a step toward a circular economy. Calcium, at ~15 mM in PG leachate, creates inorganic precipitation that interferes with biological sulfate reduction, the first step of S recovery. Here, we evaluated the effectiveness of using cation-exchange to lower the calcium concentration in water-leached PG (PG water) delivered to a H₂-based membrane biofilm reactor (H₂-MBfR) employed to reduce sulfate to sulfide. A high sulfate flux (1 gS/m²-day) and 65% sulfate reduction were achieved despite a high pH (10) resulting from base production during sulfate reduction. However, soluble sulfide was only 20% of the reduced S, possibly due to precipitation of sulfide, iron, and phosphate, and alkalinity analysis revealed possible formation of polysulfides. Shallow metagenomics of the biofilm documented that <i>Desulfomicrobium</i> was the dominant sulfate-reducing bacterium, while <i>Thauera</i>, a mixotroph capable of sulfate reduction and sulfide oxidation, also was an important genus. The metagenomics also revealed the presence of methanogens and acetogens that competed for H₂ and CO₂. Although calcium removal from PG water improved sulfate reduction and reduced inorganic precipitation in the H₂-MBfR, soluble sulfide generation must be improved by supplying sufficient CO₂ to moderate pH increase due to sulfate reduction and by controlling the H₂-delivery capacity to limit methanogens and acetogens.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 10","pages":"2701-2708"},"PeriodicalIF":3.6,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144565957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}