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Sunday
BIOT reception
07:00pm - 09:00pm USA / Canada - Eastern - August 22, 2021
Division: [BIOT] Division of Biochemical Technology
Session Type: Networking Events - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology
Upstream Processing: Microbial Metabolic Engineering: Microbial Metabolic Engineering: Bacterial Hosts
10:30am - 12:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Aditya Kunjapur, Presider, MIT; Tiffany Rau, Presider, ‍ ; Josh Leonard, Presider, ‍ ; Quinn Mitrovich, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Advances in synthetic biology have enabled the design and construction of cell factories for the cleaner production of chemicals and other biological products important to humans. Metabolic engineering aims to develop tools and strategies that can be used in the optimization of biochemical pathways and in the design and implementation of non-native pathways, leading to more efficient biocatalysts and access to novel products. For this session we welcome presentations on topics related to microbial metabolic engineering, including the design, construction and testing of whole-cell biocatalysts, the development of tools for metabolic modeling and profiling, and the incorporation of novel substrates and biochemical reactions within biological systems.

Monday
Developing a novel microbial chassis for upcycling waste polyethylene terephthalate
10:30am - 10:50am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Jinjin Diao; Yifeng Hu; Prof. Tae Seok Moon, Presenter, Washington University in St. Louis
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Polyethylene terephthalate (PET) represents 8% of global solid waste. PET chemical recycling has been an option to solve this global problem, but it suffers from its relatively high process cost and the extremely low price of virgin PET. One solution to address this issue is to upcycle waste PET rather than recycle it to generate the same PET typically with low quality. PET upcycling can be achieved by depolymerizing PET into terephthalic acid (TPA) and ethylene glycol (EG) and biologically converting these monomers into value-added products. However, there are only a handful of reports demonstrating microbial strains capable of growing on both TPA and EG generated from PET as sole carbon sources. To overcome this critical limitation, we have performed strain screening to discover a Rhodococcus strain (named RPET) that can grow well on the alkaline hydrolysis products of PET as the sole carbon source without any purification step. Notably, this strain was able to tolerate and grow on a mixture of TPA and EG at extremely high concentrations (up to 0.3M each, total 0.6M) and high osmolarity resulting from alkaline hydrolysis and pH neutralization. The resultant pH neutralized media supported RPET’s growth (up to 0.4 g dry cell weight per g PET) without any purification and sterilization step except for their dilution to make up to 0.6M of monomer concentrations. In addition, many synthetic biology tools, developed for a related species Rhodococcus opacus (1), were functional in RPET, facilitating its engineering. In this presentation, we will discuss our effort to develop this novel chassis for waste PET valorization with PET conversion into carotenoids and muconate as two demonstration products (2).
Monday
Overexpression of the licanantase lipoprotein in acidithiobacillus Ferrooxidans enhances copper sulfide bioleaching
10:50am - 11:10am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Rapid industrialization and an increasing drive towards electrification has led to high demands for metals, while high-grade ores reserves are depleting. Bioleaching is an attractive option to supply metals from low-grade ores and is environmentally and economically advantageous compared to conventional pyrometallurgy. Acidophilic Acidithiobacillus ferrooxidans cells obtain energy from the oxidation of iron or reduced sulfur compounds, and these bacteria play an important role in industrial bioleaching operations. Recently, the lipoprotein licanantase was identified from the secreted protein fractions of Acidithiobacillus sp. and its effect on enhancing the bioleaching rate of copper sulfide ores was reported. This suggests that augmented licanantase production may enhance the bioleaching efficiency of low-grade ores by A. ferrooxidans, although this has not been explored. We engineered A. ferrooxidans to overexpress the lipoprotein homologous to licanantase, examined the strain on the bioleaching of different copper sulfide ore components (chalcopyrite (CuFeS2), chalcocite (Cu2S), and covellite (CuS)), and explored the possible mechanism of improved bioleaching.
In experiments with all three copper sulfides, the engineered cells enhanced the production of extracellular polymeric substances (EPS) leading to increased biofilm formation. These results corresponded to the microscopic observation of higher cell attachment onto ore surfaces when using the engineered cells, indicating licanantase expression facilitated contact between the bacteria and mineral surfaces. Interestingly, the bioleaching efficiency of three ore components was affected by the engineered cells differently. Compared to abiotic or biotic wild type cell conditions, the copper leaching efficiency of the engineered cells was 23.1% and 52.7% higher on chalcocite and covellite, respectively, whereas it was lowered in chalcopyrite as more passivation (likely jarosite) was found on the leached material. We hypothesize that the additional biofilm formation by the licanantase expression may stimulate iron precipitation, which can lead to the passivation of the ore surfaces. These observations suggest an interesting opportunity to improve the bioleaching efficiency of copper sulfide ores by overexpression of licanantase lipoprotein which can be used to manipulate cell-to-mineral interfacial interactions.

Monday
Stepping on the gas to a circular economy: Accelerating development of carbon-negative chemical production from gas fermentation
11:10am - 11:30am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Michael Koepke, Presenter, LanzaTech
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Atmospheric CO2 has accumulated to levels unprecedented since the Pliocene Epoch (> 2.6 million years ago). Gas fermentation technology offers a path to produce impactful volumes of sustainable products from abundant, low value renewable carbon feedstocks. LanzaTech is pioneering the commercialization of a gas fermentation process that allows the continuous production of sustainable fuels, chemicals and protein from renewable carbon resources at scale. Our first commercial plant in China has produced over 60,000 tons sustainable ethanol averting the emission of over 120,000 tons of CO2. Further commercial plants are in design or under construction with the process having been demonstrated with waste gas from numerous industries and synthesis gas produced from agricultural and municipal waste sources. Commercial deployment activities will updated and partnerships with consumer-facing companies such as L’Oréal and the Mibelle Group using the ethanol as a “CarbonSmart™“ intermediate platform for the production of goods including cleaning products, plastics for packaging, and fibers for clothing will be highlighted.

The development of a comprehensive synthetic biology capability for gas fermenting organisms has further broadened product opportunities. Acetone and isopropanol are important industrial bulk and platform chemicals, exclusively produced from fossil resources today. We have developed a sustainable and commercially relevant route from abundant, low-cost waste gas feedstocks by engineering the biocatalyst. To achieve this, we constructed and screened a combinatorial biosynthetic pathway library using genes derived from a historical industrial strain collection and enzyme engineering. To optimize flux, we performed strain engineering using omics analysis, kinetic modelling, and cell-free prototyping to identify competing interactions between heterologous enzymes and native metabolism. We developed and scaled up a continuous fermentation process in an industrial pilot plant, consistently demonstrating commercial production rates. Life cycle analysis confirmed significant (>165%) greenhouse gas savings. We show that acetogens, despite living on the edge of life, can be efficient cell factories for chemicals production.

Monday
CRISPR-based transcriptional control in solventogenic Clostridium species using dFnCas12a
11:30am - 11:50am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Rochelle Carla Joseph, Presenter; Nicholas Sandoval, Tulane University
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The Clostridium genus encompasses several industrially relevant strains which are naturally capable of cellulosic and hemicellulosic biomass degradation, carbon fixation, advanced biofuel and commodity chemical production, and which show potential as anti-cancer therapeutics.
While this potential has begun to be realized industrially, widespread use of Clostridium has been stunted compared to workhorse organisms. Strain engineering with existing tools is time-consuming, labor-intensive and does not enable high-throughput screening. To optimize the use of Clostridium in industry, tools must be developed which enable rapid genotype to phenotype mapping, as well as, perturbation of gene expression for tight and predictable control of protein production.

Here we demonstrate gene repression in solventogenic Clostridium species through use of a nuclease-deactivated Francisella novicida Cas12a (dFnCas12a) based CRISPR system. The recognition of a T-rich protospacer adjacent motif (PAM) site makes dFnCas12a suited for use in organisms with A/T-rich genomes like Clostridium spp. We show the construction and use of a single-plasmid dFnCas12a CRISPRi system which utilizes a lactose inducible promoter for controlled expression of the dFnCas12a gene and a single-step golden gate assembly for the insertion of target sequences into the crRNA.

We achieve over 90% transcriptional-level repression of targeted genes in C. pasteurianum (Cpa) and C. acetobutylicum (Cac). We use our CRISPRi system to eliminate solvent production in Cac through targeted repression of spo0A gene. Metabolite analysis via liquid chromatography shows this redistribution of carbon flux in undetectable levels of butanol and acetone and increased butyrate production. We describe the simultaneous repression of multiple genes through the expression of a single multi-plexed CRISPR array. We show rapid construction of duplexed CRISPR arrays via annealed 72-nucleotide oligos, and the potential to insert any number of targets. Finally, we apply this system in Cpa to demonstrate its applicability across Clostridium species, targeting the 1,3-propanediol pathway and hydrogen production.

Monday
Portable hairpin structures at the mRNA leader region to fine-tune the expression levels in Bacillus
11:50am - 12:10pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Qin Wang, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The 5’-untranslated region (5’-UTR) of prokaryotic mRNAs plays an essential role in post-transcriptional regulation. Bacillus species, such as Bacillus subtilis and Bacillus licheniformis, have gained considerable attention as microbial cell factories for the production of various valuable chemicals and industrial proteins. We developed a portable 5’-UTR sequence for enhanced protein output in the industrial strain B. licheniformis DW2. This sequence contains only ~30-nt and forms a hairpin structure located right before the open reading frame. The optimized Shine-Dalgarno (SD) sequence was presented as a single strand on the loop of the hairpin for better ribosome recognition and recruitment. After optimizing the free energy of folding, this 5’-element could effectively enhance the expression of eGFP by ~50-fold, and showed good adaptability for other target proteins including RFP, nattokinase and keratinase. It is well-known that stable structure at a ribosome binding site (RBS) impedes translation initiation. Furthermore, by varying both the folding energy of the hairpin structures and the lengths of the spacer sequences, we generated an incremental and predictable expression library over ~100-fold range, which has great potential to be used for fine-tuning gene expression in metabolic engineering.
Monday
Implementation of CRISPR-dCas9 system in Geobacter sulfurreducens to enhance the release of fixed ammonium
12:10pm - 12:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Mark R. Poole, Presenter, North Carolina State University; Douglas Call, North Carolina State University; Amy Grunden; Rodolphe Barrangou
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Nitrogen gas (N2) conversion to ammonium (NH4+) is an essential but cost- and energy-intensive process, with the Haber-Bosch method constituting the major form of artificial N2 fixation implemented today. Biological nitrogen fixation is an alternative process that is more energy efficient, but faces significant barriers to commercial implementation including slow rates of fixed nitrogen production. Geobacter sulfurreducens is a nitrogen-fixing soil bacterium that can respire on anode electrodes. The metabolic rate of G. sulfurreducens while respiring in microbial electrochemical cells (MECs) is connected to nitrogen fixation, wherein higher applied voltages can drive higher N2 fixation rates. The cells do not secrete NH4+ during anode respiration. Tight regulatory control quickly converts NH4+ into molecules such as amino acids. Enabling excretion of NH4+ requires genetic manipulation of relevant regulatory pathways. The CRISPR-dCas9 system is a robust method for offsetting undesirable regulatory control. In this study, the CRISPR-Cas9/dCas9 system was implemented in G. sulfurreducens to encourage nitrogen release. Nitrogenase activity and ammonium uptake were targeted through the generation of various knockdown mutants. These knockdown mutants were evaluated for nitrogen fixation rate, respiration rate and ability to secrete fixed ammonium. The generation of Geobacter sulffureducens mutants capable of ammonium secretion via CRISPR-dCas9 demonstrates a valuable new genetic tool for metabolic engineering and provides a useful example of MEC-driven biochemical production.
Downstream Processing: Non-Chromatography Based Separation of Biomolecules: Non-Chromatography Based Separation of Biomolecules: mAbs
10:30am - 12:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Mahsa Rohani, Presider, ‍ ; Akshat Gupta, Presider
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Protein purification methods based on mechanical separations like centrifugation, hydro cyclones and elutriation along with depth filtration, multi-phase partitioning, precipitation, flocculation, and crystallization are widely used in biopharmaceutical industry. These techniques enable, enhance and complement many key and novel separations required for purification of biomolecules and are being actively studied and improved in order to meet evolving needs of industry and a higher demand for performance. This includes effective harvesting of higher density cell cultures; enhanced impurity clearance; enhanced performance of chromatography, sterile and virus filtration steps; in stand-alone, integrated, or continuous/semi-continuous manner. These technologies also play a pivotal role in identifying novel ways of using conventional unit operations to solve both current and future bioprocessing challenges of complex biological products. This session seeks to report advances in the development, fundamental understanding, and industrial application of non-chromatographic, non-membrane-based unit operations to achieve desired bio separations, as well as cases demonstrating the advantages/disadvantages of integrated processes thereof. Operations of interest may include; traditional unit operations, centrifugation, flocculation, depth filtration or less traditional unit operations, hydro cyclones, elutriation, acoustic separation, aqueous multi-phase partitioning, precipitation, crystallization and polymer-aided flocculation. In addition, we would like to welcome both experimental and modeling submissions. Priority will be given to those that provide insights and present approaches of general utility, and for whom experimental and/or manufacturing implementations are presented and compared with alternative approaches.

Monday
Antibody capture based on magnetic beads from culture at density > 100 x 106 cells/mL
10:30am - 10:50am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Nils Brechmann, Presenter, KTH, Royal Insitute of Technology; Hubert Schwarz; Kristofer Eriksson; Véronique Chotteau
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Cell clarification represents a major challenge in very high cell density processes for the production of biopharmaceuticals such as monoclonal antibodies (mAb). Typically, this step requires dedicated equipment, has potentially high consumable costs, is time intensive, requires dilution or addition of flocculation chemical and, importantly, lead to high host cell proteins (HCP) levels. To reduce the pressure on the downstream side, we propose a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Here, the performances of magnetic bead-based mAb capture on non-clarified cell suspension from intensified fed-batch culture at very high cell density were studied. The mAb capture from a culture in stirred tank bioreactor at density larger than 100 x 106cells/mL provided an adsorption efficiency of 99 %, an overall yield of 93 % with a logarithmic HCP clearance of ≈ 2-3 and a resulting HCP concentration ≤ ≈5 ppm. The mAb quality attributes of charge variant and aggregation were not affected. These results show that the proposed process of direct capture from very high cell density cell suspension is possible without prior processing. This technology brings significant benefits in terms of operational cost reduction, time and improvements such as low HCP. It is a powerful tool alleviating the challenge of process intensification by very high cell concentration.
Figure: Magnetic purification directly applied to the harvested cell suspension; adsorption efficiency (left panels A, C, E); total binding after 2 h and total elution yield (right panels B, D, F).

Figure: Magnetic purification directly applied to the harvested cell suspension; adsorption efficiency (left panels A, C, E); total binding after 2 h and total elution yield (right panels B, D, F).


Monday
Evaluation of different methods for enabling an efficient harvest process for monoclonal antibodies from high cell density cultures
10:50am - 11:10am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Mammalian CHO cell culture is commonly used for monoclonal antibody (mAb) production due to the ability to incorporate post-translational modifications, such as glycosylation. Despite improvements in process titers (~10g/L), long culture durations (12-14 days) still result in higher overall costs. Non-mammalian expression systems like yeast and fungal-based platforms present attractive alternatives with potential higher cell titers (~20g/L), shorter culture durations (5 days) while retaining the capability for post-translational modifications. However, these culture processes have much smaller cell sizes (3-5 um) and higher cell densities (30-40% packed cell volume (PCV)), compared to CHO cell culture processes (10 um and 5-10% PCV), presenting new challenges to the downstream clarification process where centrifugation and/or depth filtration have historically been used.
In this study, we investigate and compare different commercial and novel clarification methods for harvesting monoclonal antibodies from high cell density yeast cultures in batch or fed-batch processes, including depth filtration, pretreatment followed by depth filtration (pH, flocculation agent), body feed filtration and gravity assisted devices. Although depth filtration can deliver desired clarity (<6 NTU), it has a very limiting throughput (<20 L/m2) when used alone. Pretreatment of the feed streams with flocculation agents significantly enhanced the filtration performance by depth filters and allowed the exploration with novel devices using gravity assisted settling or barrier filtration. The addition of filter aids enabled body feed filtration with disposable units and demonstrated significant improvement in processing throughput (> 100 L/m2). Process performance (recovery, processing time, development time), economics (capital cost, cost of consumables, cost of validation) and manufacturability (ease of scale-up, robustness) are also evaluated and compared across different methods in the study when considering 1000L scale production process.

Monday
Breakthrough of process related impurities during depth filter harvest of high cell density CHO culture
11:10am - 11:30am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Miss Maria Parau, Presenter, University College London; Daniel Bracewell, UCL Dept Biochemical Engr
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Upstream developments have led to increased mAb titres above 5g/L in 14-day fed-batch cultures, however this has come with the disadvantage of cell density exceeding 20 million cells/mL. Depth filtration remains a popular choice for harvest of CHO cell culture for bioreactors up to 2000L and single use facilities. However due to the high cell density there is also increase of process related impurities, such as cell debris, host cell protein (HCP) and DNA. This can affect the capacity of the depth filters and lead to carry over of impurities to Protein A chromatography leading to early resin fouling.

New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence there is a need for process understanding of impurity removal mechanisms. In this work the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. The CHO cell culture was initially harvested using Millistak+ D0HC primary depth filter for both secondary filter conditions. The viability of the cell culture is also altered to examine its effect on depth filter performance. Additionally a novel method was developed to image the fouled depth filters under a confocal microscope where PicoGreen and Nile Red were used to stain fouled depth filters for DNA and debris, respectively.

Flux, tested at 75, 100 and 250 LMH, w as found to affect the maximal throughput but no significant changes were seen in the HCP and DNA breakthrough. However a drop in cell culture viability, from 87% to 37%, led to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability. The X0SP filter has a 30% higher maximal throughput, which can be explained by the confocal imaging where the debris and DNA is distributed differently in the layers of the filter pods, with more of the second tighter layer used in the X0SP.

Monday
Understanding prefiltration and fouling of virus filters
11:30am - 11:50am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Solomon Isu; Sumith Wickramasinghe; Andrew Zydney; Dr. Xianghong Qian, Presenter, University of Arkansas at Fayetteville
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Virus clearance is an essential component of the downstream purification of biopharmaceutical products. Virus filtration is routinely conducted towards the end of the purification train to ensure effective removal of both endogenous and adventitious virus during the manufacture of protein therapeutics. Membrane fouling and the performance of virus filters (filtration flux and throughput) could be strongly affected by product properties (charge, hydrophobicity and conformational stability), solution conditions, and the properties of the virus filter. Fouling is typically dominated by product related foulants such as soluble aggregates (dimers, trimers) insoluble aggregates, and product variants (such as denatured or mis-folded product and product(s), with markedly different post translational modifications), although other impurities such as host cell proteins (HCPs), DNA and spiked virus particles can also contribute to the overall flux decline. Use of a prefilter is often critical when developing a practical virus filtration step. The specific mechanisms for foulant removal during prefiltration include size exclusion, ion-exchange and hydrophobic interaction. Here the effects of prefilter type, with a single mechanism of action, on the filtration performance of one commercial virus filter was investigated. In addition, analytical tools have been used to identify specific foulant(s) that plug the virus filter by analysis of material eluted from the filter using mass spectroscopic tools including LC-MS, MALDI-MS, and capillary electrophoresis. Results show that size-based prefilters are largely ineffective, demonstrating that the foulants are not large aggregates. Hydrophobic interaction and cationic exchange prefilters provided significant improvements in the performance of the virus filter, suggesting that the foulants may be denatured product or product variants differing in charge due to post-translational modifications or differences in glycosylation. This was confirmed by the presence of high concentrations of product in the eluted samples and that the eluted samples have different molecular weight and charge profiles to those from the filtrate. These results provide significant insights on the nature of foulants that affect the performance of the virus filter and suggest important strategies for improving virus filtration processes.
Monday
Establishment of high throughput tools for antibody crystallization process development
11:50am - 12:10pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Historically, protein crystallization has been used for structure determination using x-ray diffraction. In addition to this traditional application, crystallization can be used in the downstream processing of monoclonal antibodies and other biological molecules. Typical therapeutic protein downstream processes can have high raw material costs, high buffer consumption, and long cycle times. Process intermediates and the drug substance are also typically held as low concentration solutions which can lead to protein instability and the need for large drug substance freezer storage space. Due to these challenges, opportunities exist for protein crystallization to be a lower cost alternative for product concentration, isolation, and impurity removal in drug substance manufacturing. Opportunities also exist within drug delivery as crystallization can be used to enable highly stable, high concentration formulations for subcutaneous delivery or to make crystalline protein suspensions with altered solubility and release properties.
Due to the large size and complexity of monoclonal antibodies, crystallization conditions are often difficult to identify. Broad vapor diffusion screens used for structure determination are needed to test thousands of crystallization conditions. Conditions identified can be difficult to translate to a scalable, batch mode reaction and a wide range of process parameters need to be screened during process development. To enable rapid antibody crystallization process development at Merck, a high throughput scale down model has been established through the use of a Tecan liquid handler. This work will describe the high throughput method development, current high throughput screening results, and comparison to scale up for three monoclonal antibodies. The workflows being established for successful translation of vapor diffusion conditions to a scalable batch mode process will also be discussed.

Monday
Impact of clarification on harvest concentration and capture chromatography
12:10pm - 12:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Philip Szymanski, Presenter, MilliporeSigma; Akshat Gupta; Dana Kinzlmaier; Santosh Rahane, MilliporeSigma
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Process intensification is considered the next step in the evolution of monoclonal antibody production. This work investigated harvest concentration via single-pass tangential flow filtration as an avenue towards capture chromatography intensification and demonstrated that clarification impacts both subsequent operations. With traditional cellulosic depth filters containing diatomaceous earth filter aid media as a benchmark, we compared the performance of synthetic media, flocculation with two different polymer flocculants, and acid pretreatment. Process improvements were realized in the way of higher filter throughput for clarification, higher achievable concentration factors during concentration, and time or resin savings during the capture step. Product yield, aggregate levels, host cell protein and host cell DNA concentrations were measured and tracked throughout the process.
Advances in Protein Engineering 2: Advances in Protein Engineering
10:30am - 12:35pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Nik Nair, Organizer; Zhe Rui, Organizer, UC Berkeley; Aditya Kunjapur, Presider, MIT; Joyce Liu, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Protein conjugates and fusions are expanding the toolkit for development of new molecules with wide-ranging applications as bioanalytical reagents and biomedical tools for disease diagnosis and therapy. Additionally, unique formulations provide non-covalent interactions with proteins that can enhance their stability, delivery, or function. As protein engineering and production platforms become increasingly sophisticated, there is a unique opportunity to capitalize on new technologies to develop molecules with complex modalities (fusions, bispecifics, dAbs, ADCs, etc.) as research tools and potential therapeutics. Further, high-throughput screening and deep molecular characterization are now enabling greater choice and complexity for protein formulation. This session will focus on cutting-edge approaches and methodologies for engineering, manufacturing, formulating, and characterizing proteins in this growing class of biologics. Examples of interest include, but are not limited to, protein-small molecule conjugates, multi-specific antibody-protein fusions, cyclized peptides, protein-polymer or protein-nanoparticle conjugates or non-covalent nano-assemblies, and macroscopic materials that incorporate a protein component. Abstracts will be prioritized that present advances in protein-bioconjugate construction, new classes of protein drugs, innovative targeting strategies, novel applications of protein conjugates and fusions, and unique processing or formulation strategies which take advantage of the biomolecular properties offered by these classes of molecules.

Monday
Introductory Remarks
10:30am - 10:35am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Monday
Engineering of a robust, high-fidelity DNA polymerase for use in NGS library amplification
10:35am - 11:15am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Many genomic tools are made possible by molecular assays that harness the diverse functionality of enzymes. The performance of these tools is currently limited by the intrinsic properties of their enzyme components. For example, preparation of next generation sequencing libraries from low-concentration samples requires accurate and robust PCR amplification. Utilizing our CodeEvolver® directed evolution platform, we engineered a proofreading thermostable DNA polymerase for NGS applications. In this presentation, we highlight the use of machine learning to improve replication fidelity, uniformity of coverage, and amplification yields. We dive into how these software tools are critical for the successful engineering of commercial products and touch on how to maximize the experimental results to make predictions.
Monday
Withdrawn
11:15am - 11:35am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Monday
Cell-free protein synthesis (CFPS) in polymer materials: Biosensing and other applications
11:35am - 11:55am USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Dr. Marilyn Lee, Presenter, US Army DEVCOM CBC
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Advances in synthetic biology have granted the ability to dictate a variety of functions through genetic and protein design. These functions include sensing, catalyzing chemical reactions, and producing desired molecules and materials. While these capabilities have been extensively developed in whole cell cultures or in vitro in solution, important applications may also be pursued for biological systems in solid materials. Challenges obstructing this concept include difficulties in maintaining cell viability or protein activity during the harsh processing steps involved in producing many synthetic materials and experienced over long term use of the material. One technology that shows promise in circumventing these issues are cell-free protein synthesis (CFPS) systems. CF systems are comprised of lysates, or broken cells, that contain all of the enzymes, DNA, and active components of a cell. These components can be reactivated and supplied with resources to carry out sensing or catalysis without the burden of maintaining cellular survival and replication. CF reactions may be dried and stored for months to a year and maintain activity upon rehydration. Further, dried CF reactions have shown remarkable stability to both heat and organic solvents lending them to applications in synthetic materials that would be impossible using whole living cells. These characteristics have inspired us to reconfigure the reaction environment and embed cell-free systems in polymer matrices towards enabling new form factors and controlling water-induced activation.

Monday
Stabilization of a heterotrimeric FAD dependent glucose dehydrogenase by introducing inter-subunit disulfide bonds
11:55am - 12:15pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
In this study, the stabilization of heterotrimeric FAD dependent glucose dehydrogenase (FADGDH) is reported, which was achieved by designing and introducing inter-subunit disulfide bonds.
The direct electron transfer (DET) type FADGDH is a heterotrimeric enzyme, which is composed of three subunits; a hitchhiker protein (γ), a catalytic subunit harboring FAD/3Fe4S cluster (α), and a three heme cs containing electron transfer subunit (β). This heterotrimeric structure is very unique among GMC oxidoreductase family. The presence of electron transfer subunit makes this enzyme capable to transfer electron directly to the electrode which is recognize as a DET-type enzyme.
Our former achievement revealed that the γα complex structure (PDB:6a2u) harbors the intrinsic disulfide bond between α and γ subunit, which contributes the stabilization of γα complex. Besides, DET type FADGDH showed two phase thermal inactivation, where the first phase is due to the dissociation of β subunit from γαβ complex. This was confirmed by the chemical crosslinking of γαβ complex with glutaraldehyde brought the high thermal stability. Therefore, the stability of this heterotrimeric enzyme is strongly dependent on the interaction of β subunit with γα complex. These observations encouraged us to introduce the inter-subunit disulfide bonds to improve the stability of this enzyme.
In this study, based on recently elucidated partial structure of DET type GDH heterotrimer complex, amino acid substitutions were designed to form the inter-subunit disulfide bonds. From the constructed several mutants, we identified the positions where Cys residues should be introduced. As the results, mutant with newly introduced cysteine residues were recombinantly prepared as the soluble heterotrimer complex, without losing its catalytic activity. The formation of disulfide bonds were confirmed by SDS-PAGE analyses by comparing the migrated bands before and after the reduction of protein samples. Thus designed and prepared Cys-substituted FADGDH showed drastic increase in the thermal stability, which was not inactivated under 65 °C of incubation for more than 30 min. Electrochemical investigation suggested that the Cys-substituted FADGDH kept its characteristic DET ability to the electrode, indicating that thus designed engineered DET type FADGDH gives promise the future application for the continuous glucose monitoring system.

Monday
Biomineralization by design: Application of de novo proteins for nanocrystal synthesis
12:15pm - 12:35pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Biomineralization – the synthesis of inorganic materials using proteins – has recently gained interest as a low cost, green route for the production of metal chalcogenide semiconductor nanocrystals. Typical biomineralization techniques use one of two methods to either catalyze or template the reaction: one type uses enzymes to drive the reaction by producing a reactive chalcogenide species, while the other type uses metal-binding proteins to template the crystallization of a chemical-based synthesis. An idealized biomineralization approach would use proteins with both functionalities, creating a unified system for catalyzing and controlling nanocrystal growth; however, nature has yet to provide such a system. Herein, we demonstrate the biomineralization of metal chalcogenide nanocrystals using a newly synthesized de novo protein, ConK, capable of both catalyzing and templating nanocrystals during growth. Made entirely by design, de novo proteins are highly stable and tolerant to mutations in the amino acid sequence, allowing facile modification and the introduction of new protein functionalities. We created a collection of modified ConK proteins which produce well controlled nanocrystal populations of various size. We characterized the optical properties of the resultant nanocrystal populations using absorbance, fluorescence, and transient absorbance spectroscopy. We verified the crystal structure and nanocrystal size using XRD and TEM measurements. Importantly, semiconductor nanocrystals synthesized using ConK demonstrate improved fluorescence and stability compared to those obtained using naturally derived biomineralization pathways, making de novo biomineralization ideal for commercial implementation.
Upstream Processing: Microbial Metabolic Engineering: Microbial Metabolic Engineering: Eukaryotic Hosts
02:00pm - 04:00pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Aditya Kunjapur, Presider, MIT; Josh Leonard, Presider, ‍ ; Tiffany Rau, Presider, ‍ ; Quinn Mitrovich, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Advances in synthetic biology have enabled the design and construction of cell factories for the cleaner production of chemicals and other biological products important to humans. Metabolic engineering aims to develop tools and strategies that can be used in the optimization of biochemical pathways and in the design and implementation of non-native pathways, leading to more efficient biocatalysts and access to novel products. For this session we welcome presentations on topics related to microbial metabolic engineering, including the design, construction and testing of whole-cell biocatalysts, the development of tools for metabolic modeling and profiling, and the incorporation of novel substrates and biochemical reactions within biological systems.

Monday
Reducing rare codon content in heterologous anaerobic fungal genes reveals efficient mevalonate pathway homologs
02:00pm - 02:20pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Anaerobic fungi are an emerging platform for biotechnology with prolific plant biomass degrading capabilities and novel biosynthetic pathways. Yet, heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find that one reason is that the genome composition of anaerobic fungi, like P. indianae, is extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli. While codon biases are broadly predicted by standard codon adaptation index (CAI) metrics, the overreliance on specific codons are not explicitly captured. Native P. indianae genes with these extreme biases create significant growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs but less dependence on such rare codons. However, codon optimization rescues growth allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway increasing production of mevalonate up to 2.5-fold and reducing carbon loss to acetate by ~90% under the conditions tested. Our work highlights the need for in depth codon utilization analysis in determining heterologous hosts for expression and demonstrates the potential for anaerobic fungal enzyme homologs in metabolic engineering.
Monday
Reaction compartmentalization leads to enhanced production of PHA in Yarrowia lipolytica
02:20pm - 02:40pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Making the most efficient use of the finite amount of carbon and energy present in feedstocks is a common goal for the microbial production of commodity chemicals, thus improving the yield of reactions and improving process economics. A well-studied method to improve reaction efficiency is to increase the local concentration of the reactants. To this end, we have focused on increasing the efficiency of reactions building off the beta-oxidation cycle of Yarrowia lipolytica as a means of improving production of the bioplastic polyhydroxyalkanoate (PHA). Local concentration changes are achieved by compartmentalizing key enzymes within the peroxisome, a membrane-enclosed organelle where beta-oxidation occurs. Import into this organelle is accomplished through recognition of some form of peroxisomal targeting signal (PTS) and mediated by a cascade of peroxisome-associated (PEX) enzymes. Manipulation of this tag and the associated enzymes resulted in a greater than three-fold increase in PHA production from the same carbon feed conditions. In an effort to further improve production, expansion of the total peroxisome volume was investigated through knockout and overexpression of specific PEX enzymes. Finally, improvements in carbon flux into the peroxisome were sought via traditional metabolic engineering strategies including carbon-sink knockouts, transporter manipulation, and pathway overexpression.
Monday
Genome-wide knockout screens reveal new engineering targets that enhance protein secretion in a non-model yeast
02:40pm - 03:00pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The yeast Komagataella phaffii (Pichia pastoris) is commonly used for the production of recombinant proteins due to its rapid growth to high cell densities and its ability to secrete proteins into the culture medium. Large-scale clinical manufacturers routinely achieve industrially relevant titers for protein therapeutics like insulin and subunit vaccines including for the COVID-19 pandemic. There is growing interest in K. phaffii as an alternative host for the manufacturing of more complex, high-value protein therapeutics like monoclonal antibodies (mAbs), including several recent product approvals. Early engineering efforts yielded humanized yeast strains capable of secreting mAbs with specific quality attributes and post-translational modifications. Secreted titers must be increased, however, to realize cost-saving or scalability advantages over current hosts like CHO cells.

While integration of exogenous genes can confer new cellular functions and post-translational modifications, engineering of the endogenous host-cell genome may expand overall secretory capacity through deletion of unneeded proteins and pathways or expansion of secretory organelles. In a non-model host like K. phaffii, only 50-70% of the genome is functionally annotated, limiting the extent of rational genomic engineering. Tools for unbiased functional genomics are needed to discover new engineering targets that may improve protein secretion.

Here, we present the application of two CRISPR-Cas9 knockout libraries for the discovery of new engineering targets in K. phaffii. First, we targeted all endogenous secreted host cell proteins and identified non-essential genes in the yeast secretory pathway. Deletion of ~10 non-essential HCPs yielded strains with increased secreted titer and purity of several recombinant proteins. Second, we created a pooled genome-wide knockout library. We employed a novel flow-cytometric assay to identify library members with enhanced secretion of a mAb. Subsequent validation of discovered knockouts yielded new targets for strain engineering. These unbiased approaches to serve as powerful demonstrations for the genomic engineering of poorly characterized manufacturing hosts for complex phenotypes.

Monday
Developing genetic tools for Methylomicrobium album BG8: A versatile microbial platform for conversion of methane to isoprenoids
03:00pm - 03:20pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Methanotrophic bacteria use methane, a greenhouse gas and cheap carbon feedstock, as a sole source of carbon and energy, and can convert it to value-added chemicals like isoprenoids which has generated interest as many of them are precursors of ultraperformance fuels, such as jet fuel. Methylomicrobium album BG8 is a methanotrophic bacterium notable for its fast and robust growth and rich genetic potential, making it an excellent candidate for development of a microbial bioproduction platform. Production of isoprenoid from methane would require controlled expression of the Methyl Erythritol Phosphate [MEP] pathway involved in the production of isopentenyl pyrophosphate [IPP] and dimethylallyl pyrophosphate [DMAPP] in M.album BG8 , two precursors of all isoprenoids. In this context, development of functional genetic tools can enable strategies for increased productivity of isoprenoids. We determined broad host range plasmids of IncP, IncQ and pBBR1 family can be conjugated and propagated successfully in M.album BG8. We tested four constitutive promoters PMMO (Particulate methane monooxygenase), PMMA (Mxa type methanol dehydrogenase), PGAP (Glyceraldehyde 3-phosphate dehydrogenase) and PPYC (Pyruvate carboxylase) and two inducible promoters PXOX (Xox type methanol dehydrogenase induced with Lanthanum) and PTet (Tetracycline promoter) using GFP expression. GFP expression data shows PMMO and PMMA to be high expression while PGAP to be a low expression promoter. Of the two inducible promoters PTet shows tightly controlled expression while Pxox shows to be partially inducible. We validate the kit by controlled expression of an esterase under PMMO and PGAP. This work will lay the basis for use of M. album BG8 as a microbial platform suitable for industrial conversion of methane to a vast array of isoprenoids of interest.
Characterization of different promoters via GFP expression in <i>M.album</i> BG8

Characterization of different promoters via GFP expression in M.album BG8


Monday
Low temperature crippled the distributed metabolism of partial nitritation/anammox (PN/A) reactor
03:20pm - 03:40pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Ananda Bhattacharjee, Presenter; Bishav Bhattarai, Presenter; Sha Wu; Soklida Hong, University of Utah; Ramesh Goel, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

The partial nitritation (PN) and anaerobic ammonia oxidation (anammox) process is an energy-efficient innovative biotechnological revolution for nitrogen removal from wastewaters. The high-stress susceptibility of PN and anammox process towards fluctuations in temperature is an impediment for full-scale deployment under mainstream conditions. Here, we investigate the response of microbial biofilm for PN-anammox activity to temperature gradients (35°C, 21°C & 13°C). The average NH4+-N removal declined with a decrease in temperature. At 13°C, the PN-anammox process was deemed unstable with high nitrite (>20mgN L-1). The genes and transcripts of metagenome-assembled genomes (MAGs) were used to investigate the stress response of PN and anammox process. Briefly, a 9-L batch PN/A reactor, fed with reject water from anaerobically digested sludge, was exposed to temperature gradients of 35°C, 21°C & 13°C. RNA & DNA were extracted from biomass collected at each gradient post acclimatization period. The DNA sequences were assembled into contigs using SPAdes & subsequently binned into MAGs using MetaBAT. The RNA-Seq data were mapped to the gene of MAGs with BBMap. Sixty-seven MAGs were recovered from the PN-anammox reactors. The community-wide expression of genes differed with the different temperatures. The specific activity and transcriptional response of ammonia-oxidizing bacteria and anammox bacteria declined at low temperatures. At 13°C, low temperature, the number of MAG genes expressed reduced by three-folds, crippling the partial nitritation and anammox process. The genome-based analysis reveals that anammox species and other vital microbes differ in their gene expression responses to different temperatures. The metabolic pathways inferred from MAGs indicate that PN-anammox bioreactor microbes are metabolically versatile and exhibited novel opportunities for metabolic cooperation.

Monday
Changes in intracellular enzyme ratios enable fine-tuning of fatty acid profiles in Escherichia coli
03:40pm - 04:00pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Kathryn Mains, Presenter, University of Colorado, Boulder; Jerome Fox, University of Colorado, Boulder
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Microorganisms continue to gain traction as viable hosts for overproducing free fatty acids, which are useful starting points for building renewable oleochemicals. To date, most studies have focused on engineering native or heterologous enzymes to adjust fatty acid production in genetically tractable hosts. This work explores kinetically guided approaches for making similar adjustments without new enzymes. In brief, we combined a detailed kinetic model of the E. coli fatty acid synthase (FAS), a fully reconstituted in vitro system, and several engineered strains to investigate the influence of changes in the ratios of four native FAS components (i.e., β-ketoacyl-ACP synthases [KAS] I, II and III and a thioesterase) on FAS outputs. At the outset of the project, our in silico and in vitro analyses suggested that high levels of ‘TesA and low levels of KAS I and II, which compete for acyl-acyl carrier protein (ACP) substrates, could reduce the average length of fatty acids, while the opposite ratio increases length. We applied this “ratiometric” tuning strategy in vivo by using a multi-plasmid system to overexpress or inhibit the expression of target enzymes; indeed, changes in FAS compositions could shift profiles toward shorter or longer chains. Intriguingly, strategies for reducing chain lengths (which included overexpression of FabH and/or CRISPRi-mediated depletion of FabF or FabB) were more limited, perhaps, as a result of the toxicity of these adjustments to growing cells. Ultimately, this work provides a kinetically guided approach (one highly complementary to protein engineering efforts) for fine-tuning the titer and product distribution of FASs in engineered strains.