2-Stage microfermentations.

Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of protocols for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, by detailing the approach to protocol development, these methods are also adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.

Effects of a smartphone application named "Shared Decision Making Assistant" for informed patients with primary liver cancer in decision-making in China: a quasi-experimental study.

BACKGROUND: It is well known that decision aids can promote patients' participation in decision-making, increase patients' decision preparation and reduce decision conflict. The goal of this study is to explore the effects of a "Shared Decision Making Assistant" smartphone application on the decision-making of informed patients with Primary Liver Cancer (PLC) in China. METHODS: In this quasi-experimental study , 180 PLC patients who knew their real diagnoses in the Eastern Hepatobiliary Surgery Hospital, Naval Medical University, Shanghai, China, from April to December 2020 were randomly assigned to a control group and an intervention group. Patients in the intervention group had an access to the "Shared Decision Making Assistant" application in decision-making, which included primary liver cancer treatment knowledge, decision aids path, continuing nursing care video clips, latest information browsing and interactive platforms. The study used decision conflict scores to evaluate the primary outcome, and the data of decision preparation, decision self-efficacy, decision satisfaction and regret, and knowledge of PLC treatment for secondary outcomes. Then, the data were entered into the SPSS 22.0 software and were analyzed by descriptive statistics, Chi-square, independent t-test, paired t-test, and Mann-Whitney tests. RESULTS: Informed PLC patients in the intervention group ("SDM Assistant" group) had significantly lower decision conflict scores than those in the control group. ("SDM Assistant" group: 16.89 ± 8.80 vs. control group: 26.75 ± 9.79, P < 0.05). Meanwhile, the decision preparation score (80.73 ± 8.16), decision self-efficacy score (87.75 ± 6.87), decision satisfaction score (25.68 ± 2.10) and knowledge of PLC treatment score (14.52 ± 1.91) of the intervention group were significantly higher than those of the control group patients (P < 0.05) at the end of the study. However, the scores of "regret of decision making" between the two groups had no statistical significance after 3 months (P > 0.05). CONCLUSIONS: Access to the "Shared Decision Making Assistant" enhanced the PLC patients' performance and improved their quality of decision making in the areas of decision conflict, decision preparation, decision self-efficacy, knowledge of PLC treatment and satisfaction. Therefore, we recommend promoting and updating the "Shared Decision Making Assistant" in clinical employment and future studies.

"Multiagent" Screening Improves Directed Enzyme Evolution by Identifying Epistatic Mutations.

Enzyme evolution has enabled numerous advances in biotechnology and synthetic biology, yet still requires many iterative rounds of screening to identify optimal mutant sequences. This is due to the sparsity of the fitness landscape, which is caused by epistatic mutations that only offer improvements when combined with other mutations. We report an approach that incorporates diverse substrate analogues in the screening process, where multiple substrates act like multiple agents navigating the fitness landscape, identifying epistatic mutant residues without a need for testing the entire combinatorial search space. We initially validate this approach by engineering a malonyl-CoA synthetase and identify numerous epistatic mutations improving activity for several diverse substrates. The majority of these mutations would have been missed upon screening for a single substrate alone. We expect that this approach can accelerate a wide array of enzyme engineering programs.

Two-stage dynamic deregulation of metabolism improves process robustness & scalability in engineered E. coli.

We report that two-stage dynamic control improves bioprocess robustness as a result of the dynamic deregulation of central metabolism. Dynamic control is implemented during stationary phase using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. Deregulated networks are less sensitive to environmental conditions improving process robustness. Process robustness in turn leads to predictable scalability, minimizing the need for traditional process optimization. We validate process robustness and scalability of strains and bioprocesses synthesizing the important industrial chemicals alanine, citramalate and xylitol. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology.

"Shared Decision Making Assistant": A Smartphone Application to Meet the Decision-Making Needs of Patients With Primary Liver Cancer.

The development of medical technology provides medical specialists with a variety of choices for their primary liver cancer patients, including partial liver resection, transcatheter arterial chemoembolization, liver transplantation, and so on. However, in this context, because patients with primary liver cancer frequently do not receive adequate information to help make complicated medical decisions, those patients, who are usually otherwise ignorant about their disease, are facing multiple difficult choices. The problem might be alleviated with a process called "shared decision making." Accordingly, researchers developed a smartphone application named "Shared Decision Making Assistant" for primary liver cancer patients in China, and in this article, we report the process of its development. First, individual interviews were conducted to identify the specific needs and status of primary liver cancer patients participating in shared decision making. Next, expert group discussions were held among primary liver cancer medical experts, nurses, and software engineers, using a decision-making process called the Delphi method, which was used to arrive at a group opinion or decision by surveying a panel of experts, to draft the framework and decide on the contents of the mobile health-based decision aids program. Feedbacks and suggestions were collected to optimize the workflow of "Shared Decision Making Assistant." The resulting application consisted of seven modules: personal information, primary liver cancer treatment knowledge center, decision aids path, continuing care, interactive platform, health education, and backstage management.

Dynamic control over feedback regulatory mechanisms improves NADPH flux and xylitol biosynthesis in engineered E. coli.

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways.

Escherichia coli Cas1/2 Endonuclease Complex Modifies Self-Targeting CRISPR/Cascade Spacers Reducing Silencing Guide Stability.

CRISPR-based interference has become common in various applications from genetic circuits to dynamic metabolic control. In E. coli, the native CRISPR Cascade system can be utilized for silencing by deletion of the cas3 nuclease along with expression of guide RNA arrays, where multiple genes can be silenced from a single transcript. We notice the loss of spacer sequences from guide arrays utilized for dynamic silencing. We report that unstable guide arrays are due to expression of the Cas1/2 endonuclease complex. We propose a model wherein basal Cas1/2 endonuclease activity results in the loss of spacers from guide arrays. Subsequently, mutant guide arrays can be amplified through selection. Replacing a constitutive promoter driving Cascade complex expression with a tightly controlled inducible promoter improves guide array stability, while minimizing leaky gene silencing. Additionally, these results demonstrate the potential of Cas1/2 mediated guide deletion as a mechanism to avoid CRISPR based autoimmunity.

Media Robustness and Scalability of Phosphate Regulated Promoters Useful for Two-Stage Autoinduction in E. coli.

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the evaluation of the media robustness of a small library of E. coli PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media. Expression was both media and promoter dependent. Of the 16 promoters tested, 4 were identified to have tightly controlled expression, which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. This subset of PhoB activated promoters, useful for two-stage autoinduction, highlights the impact of the environment on the performance of biological parts and the importance of robustness testing in synthetic biology.

Scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion.

We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed-batch fermentations enables cell densities of ∼30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 µl (384-well plates), 100 µl (96-well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ∼5-7 gCDW/L, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.

Mapping a ketosynthase:acyl carrier protein binding interface via unnatural amino acid-mediated photo-cross-linking.

Probing and interrogating protein interactions that involve acyl carrier proteins (ACP's) in fatty acid synthases and polyketide synthases are critical to understanding the molecular basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-cross-linking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-cross-linking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACP's, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addition, FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-cross-linking. Furthermore, the ability to install the photo-cross-linking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in determining the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the molecular details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-cross-linking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.

Reprogramming acyl carrier protein interactions of an Acyl-CoA promiscuous trans-acyltransferase.

Protein interactions between acyl carrier proteins (ACPs) and trans-acting acyltransferase domains (trans-ATs) are critical for regioselective extender unit installation by many polyketide synthases, yet little is known regarding the specificity of these interactions, particularly for trans-ATs with unusual extender unit specificities. Currently, the best-studied trans-AT with nonmalonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed an assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a noncognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions and for engineering modular polyketide synthases to produce analogs.

Congruence in symptom assessment between hepatocellular carcinoma patients and their primary family caregivers in China.

OBJECTIVES: The aims of this cross-sectional study were to explore the agreement in symptom evaluation results between patients and their family caregivers and to search for the possible factors influencing the agreement. METHODS: A convenience sample of 280 dyads consisting of hepatocellular carcinoma patients and their family caregivers was included in this study. All of them completed the symptom checklist of Chinese version of the M. D. Anderson symptom inventory and the evaluations of six common symptoms of hepatocellular carcinoma. RESULTS: The levels of agreement ranged from moderate to substantial. A number of factors associated with caregivers (particularly depression state, age, others helping to care for the patient or not, and the relationship with patient) and patients (traditional Chinese medicine treatment, religion, KPS scores, and educational levels) were significantly correlated with levels of disparity on some symptoms. CONCLUSION: The study illustrates that family caregivers of hepatocellular carcinoma patients can provide reasonable reports on patients' symptoms. Healthcare providers need to pay special and sufficient attention to the caregivers' depression.

Symptom clusters and symptom interference of HCC patients undergoing TACE: a cross-sectional study in China.

OBJECTIVE: The aim of this study was to investigate the symptom and symptom clusters of patients with hepatocellular carcinoma (HCC) before and after transcatheter arterial chemoembolization (TACE), and to discuss the relationship between symptoms, symptom clusters, and symptom interference. MATERIALS AND METHODS: Patients with HCC who received TACE were asked to rate their symptoms using the M. D. Anderson Symptom Inventory and the symptom checklist particularly for HCC. To determine the interrelationships among symptoms and identify the symptom clusters, a principal component analysis with varimax rotation was carried out on the symptom items. Spearman correlation analysis was done to assess the relationship between symptom clusters and symptom interference. RESULTS: A total of 155 patients finished the whole procedure between November 2010 and May 2011. Before TACE, the five most severe symptoms, ranked in order, were fatigue (3.40 ± 2.26), distress (3.35 ± 2.60), sadness (3.01 ± 2.66), sleep disturbance (2.63 ± 2.57), and lack of appetite (2.26 ± 2.38). After TACE, fatigue (4.88 ± 2.31) was the most serious symptom, followed by sleep disturbance (4.80 ± 2.25), distress (4.59 ± 2.32), sadness (4.45 ± 2.16), lack of appetite (4.25 ± 2.51). Two symptom clusters were found before TACE: psychological symptom cluster and sickness symptom cluster. Two new symptom clusters were found after TACE: upper gastrointestinal symptom cluster and liver function impairment symptom cluster, with the two original symptom clusters remained relatively stable. The highest symptom interference items pre- and post-TACE were work and enjoyment of life, followed by mood. The symptoms of distress, sadness, fatigue, sleep disturbance, and lack of appetite were all significantly associated with the total interference (r = 0.443-0.615, p < 0.01 or p < 0.05). Symptom clusters were significantly correlated with the total symptom interference before and after TACE (r = 0.176-0.638, p < 0.01 or p < 0.05). CONCLUSION: Psychological symptom cluster and sickness symptom cluster are common for HCC patients before and after TACE. Liver function impairment and upper gastrointestinal symptom clusters are directly related to TACE treatment. Both the symptoms and symptom clusters have significant interference on the daily life of HCC patients undergoing TACE. However, more work is needed to further clarify the symptom clusters associated with TACE and to test the effectiveness of TACE in easing symptoms and improving quality of life of HCC patients.

A photocrosslinking assay for reporting protein interactions in polyketide and fatty acid synthases.

Understanding protein-protein interactions that occur between ACP and KS domains of polyketide synthases and fatty acid synthases is critical to improving the scope and efficiency of combinatorial biosynthesis efforts aimed at producing non-natural polyketides. Here, we report a facile strategy for rapidly reporting such ACP-KS interactions based on the incorporation of an amino acid with photocrosslinking functionality. Crucially, this photocrosslinking strategy can be applied to any polyketide or fatty acid synthase regardless of substrate specificity, and can be adapted to a high-throughput format for directed evolution studies.