Engineering carbon source division of labor for efficient α-carotene production in Corynebacterium glutamicum.

Effective utilization of glucose, xylose, and acetate, common carbon sources in lignocellulose hydrolysate, can boost biomanufacturing economics. However, carbon leaks into biomass biosynthesis pathways instead of the intended target product remain to be optimized. This study aimed to enhance α-carotene production by optimizing glucose, xylose, and acetate utilization in a high-efficiency Corynebacterium glutamicum cell factory. Heterologous xylose pathway expression in C. glutamicum resulted in strain m4, exhibiting a two-fold increase in α-carotene production from xylose compared to glucose. Xylose utilization was found to boost the biosynthesis of pyruvate and acetyl-CoA, essential precursors for carotenoid biosynthesis. Additionally, metabolic engineering including pck, pyc, ppc, and aceE deletion, completely disrupted the metabolic connection between glycolysis and the TCA cycle, further enhancing α-carotene production. This strategic intervention directed glucose and xylose primarily towards target chemical production, while acetate supplied essential metabolites for cell growth recovery. The engineered strain C. glutamicum m8 achieved 30 mg/g α-carotene, 67% higher than strain m4. In fed-batch fermentation, strain m8 produced 1802 mg/L of α-carotene, marking the highest titer reported to date in microbial fermentation. Moreover, it exhibited excellent performance in authentic lignocellulosic hydrolysate, producing 216 mg/L α-carotene, 1.45 times higher than the initial strain (m4). These labor-division strategies significantly contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources.

Structure and specificity of an anti-chloramphenicol single domain antibody for detection of amphenicol residues.

Antibiotics in aquaculture prevent bacterial infection of fish, but their misuse is a public health risk and contributes to the unintentional creation of multiresistant pathogens. Regulatory agencies cannot do the rigorous, expensive testing required to keep up with the volume of seafood shipments. Current rapid test kits for these drugs enable the increase in testing needed for adequate monitoring of food supply chains, but they lack a high degree of accuracy. To combat this, we set out to discover and engineer single-domain antibodies (VHHs) that bind to small molecule antibiotics, and that can be used in rapid test kits. The small size, solubility, and stability of VHHs are useful properties that can improve the reliability and shelf-life of test kits for these adulterants. Here, we report a novel anti-chloramphenicol VHH (Chl-VHH) with a disassociation constant of 57 nM. This was achieved by immunizing a llama against a chloramphenicol-keyhole limpet hemocyanin (KLH) conjugate and screening for high affinity binders through phage display. The crystal structure of the bound-VHH to chloramphenicol was key to identifying a mutation in the binding pocket that resulted in a 16-fold improvement in binding affinity. In addition, the structure provides new insights into VHH-hapten interactions that can guide future engineering of VHHs against additional targets.

Heterologous production of α-Carotene in Corynebacterium glutamicum using a multi-copy chromosomal integration method.

The carotenoid, α-carotene, is very beneficial for human health and wellness, but microbial production of this compound is notoriously difficult, due to the asymmetric rings on either end of its terpenoid backbone. Here, we report for the first time the efficient production of α-carotene in the industrial bacterium Corynebaterium glutamicum by using a combined pathway engineering approach including evaluation of the performance of different cyclases and analysis of key metabolic intermediates to determine flux bottlenecks in the carotenoid biosynthesis pathway. A multi-copy chromosomal integration method was pivotal in achieving stable expression of the cyclases. In fed-batch fermentation, 1,054 mg/L of α-carotene was produced by the best strain, which is the highest reported titer achieved in microbial fermentation. The success of increased α-carotene production suggests that the multi-copy chromosomal integration method can be a useful metabolic engineering tool for overexpression of key enzymes in C. glutamicum and other bacterium as well.

Optimizing recombineering in Corynebacterium glutamicum.

Owing to the increasing demand for amino acids and valuable commodities that can be produced by Corynebacterium glutamicum, there is a pressing need for new rapid genome engineering tools that improve the speed and efficiency of genomic insertions, deletions, and mutations. Recombineering using the λ Red system in Escherichia coli has proven very successful at genetically modifying this organism in a quick and efficient manner, suggesting that optimizing a recombineering system for C. glutamicum will also improve the speed for genomic modifications. Here, we maximized the recombineering efficiency in C. glutamicum by testing the efficacy of seven different recombinase/exonuclease pairs for integrating single-stranded DNA and double-stranded DNA (dsDNA) into the genome. By optimizing the homologous arm length and the amount of dsDNA transformed, as well as eliminating codon bias, a dsDNA recombineering efficiency of 13,250 transformed colonies/109 viable cells was achieved, the highest efficiency currently reported in the literature. Using this optimized system, over 40,000 bp could be deleted in one transformation step. This recombineering strategy will greatly improve the speed of genetic modifications in C. glutamicum and assist other systems, such as clustered regularly interspaced short palindromic repeats and multiplexed automated genome engineering, in improving targeted genome editing.

Modular engineering for microbial production of carotenoids.

There is an increasing demand for carotenoids due to their applications in the food, flavor, pharmaceutical and feed industries, however, the extraction and synthesis of these compounds can be expensive and technically challenging. Microbial production of carotenoids provides an attractive alternative to the negative environmental impacts and cost of chemical synthesis or direct extraction from plants. Metabolic engineering and synthetic biology approaches have been widely utilized to reconstruct and optimize pathways for carotenoid overproduction in microorganisms. This review summarizes the current advances in microbial engineering for carotenoid production and divides the carotenoid biosynthesis building blocks into four distinct metabolic modules: 1) central carbon metabolism, 2) cofactor metabolism, 3) isoprene supplement metabolism and 4) carotenoid biosynthesis. These four modules focus on redirecting carbon flux and optimizing cofactor supplements for isoprene precursors needed for carotenoid synthesis. Future perspectives are also discussed to provide insights into microbial engineering principles for overproduction of carotenoids.

Microfluidic Device to Quantify the Behavior of Therapeutic Bacteria in Three-Dimensional Tumor Tissue.

Microfluidic devices enable precise quantification of the interactions between anti-cancer bacteria and tumor tissue. Direct observation of bacterial movement and gene expression in tissue is difficult with either monolayers of cells or tumor-bearing mice. Quantification of these interactions is necessary to understand the inherent mechanisms of bacterial targeting and to develop modified organisms with enhanced therapeutic properties. Here we describe the procedures for designing, printing, and assembling microfluidic tumor-on-a-chip devices. We also describe the procedures for inserting three-dimensional tumor-cell masses, exposure to bacteria, and analyzing the resultant images.

Potent and tumor specific: arming bacteria with therapeutic proteins.

Bacteria are perfect vessels for targeted cancer therapy. Conventional chemotherapy is limited by passive diffusion, and systemic administration causes severe side effects. Bacteria can overcome these obstacles by delivering therapeutic proteins specifically to tumors. Bacteria have been modified to produce proteins that directly kill cells, induce apoptosis via signaling pathways, and stimulate the immune system. These three modes of bacterial treatment have all been shown to reduce tumor growth in animal models. Bacteria have also been designed to convert nontoxic prodrugs to active therapeutic compounds. The ease of genetic manipulation enables creation of arrays of bacteria that release many new protein drugs. This versatility will allow targeting of multiple cancer pathways and will establish a platform for individualized cancer medicine.

Quorum-sensing Salmonella selectively trigger protein expression within tumors.

Salmonella that secrete anticancer proteins have the potential to eliminate tumors, but nonspecific expression causes damage to healthy tissue. We hypothesize that Salmonella, integrated with a density-dependent switch, would only express proteins in tightly packed colonies within tumors. To test this hypothesis, we cloned the lux quorum-sensing (QS) system and a GFP reporter into nonpathogenic Salmonella. Fluorescence and bacterial density were measured in culture and in a tumor-on-a-chip device to determine the critical density necessary to initiate expression. QS Salmonella were injected into 4T1 tumor-bearing mice to quantify GFP expression in vivo using immunofluorescence. At densities below 0.6 × 10(10) cfu/g in tumors, less than 3% of QS Salmonella expressed GFP. Above densities of 4.2 × 10(10) cfu/g, QS Salmonella had similar expression levels to constitutive controls. GFP expression by QS colonies was dependent upon the distance to neighboring bacteria. No colonies expressed GFP when the average distance to neighbors was greater than 155 µm. Calculations of autoinducer concentrations showed that expression was sigmoidally dependent on density and inversely dependent on average radial distance. Based on bacterial counts from excised tissue, the liver density (0.0079 × 10(10) cfu/g) was less than the critical density (0.11 × 10(10) cfu/g) necessary to initiate expression. QS Salmonella are a promising tool for cancer treatment that will target drugs to tumors while preventing damage to healthy tissue.

Bacterial delivery of Staphylococcus aureus α-hemolysin causes regression and necrosis in murine tumors.

Bacterial therapies, designed to manufacture therapeutic proteins directly within tumors, could eliminate cancers that are resistant to other therapies. To be effective, a payload protein must be secreted, diffuse through tissue, and efficiently kill cancer cells. To date, these properties have not been shown for a single protein. The gene for Staphylococcus aureus α-hemolysin (SAH), a pore-forming protein, was cloned into Escherichia coli. These bacteria were injected into tumor-bearing mice and volume was measured over time. The location of SAH relative to necrosis and bacterial colonies was determined by immunohistochemistry. In culture, SAH was released and killed 93% of cancer cells in 24 hours. Injection of SAH-producing bacteria reduced viable tissue to 9% of the original tumor volume. By inducing cell death, SAH moved the boundary of necrosis toward the tumor edge. SAH diffused 6.8 ± 0.3 µm into tissue, which increased the volume of affected tissue from 48.6 to 3,120 µm(3). A mathematical model of molecular transport predicted that SAH efficacy is primarily dependent on colony size and the rate of protein production. As a payload protein, SAH will enable effective bacterial therapy because of its ability to diffuse in tissue, kill cells, and expand tumor necrosis.

Identification of Staphylococcus aureus α-hemolysin as a protein drug that is secreted by anticancer bacteria and rapidly kills cancer cells.

Targeted bacterial delivery of anticancer proteins has the ability to overcome therapeutic resistance in tumors that limits the efficacy of chemotherapeutics. The ability of bacteria to specifically target tumors allows for delivery of aggressive proteins that directly kill cancer cells and cannot be administered systemically. However, few proteins have been tested for this purpose. To identify effective molecules, we systematically sorted proteins that have been shown to cause mammalian cell death. The genes for five proteins were selected and cloned into Escherichia coli and Salmonella. Supernatant from cultures of the transformed bacteria was applied to flasks of MCF-7 mammary carcinoma cells to identify proteins that (1) were expressed, (2) secreted, and (3) rapidly killed cancer cells. Time-lapse images were taken to visualize mammalian cell morphology. Of the investigated proteins, α-hemolysin from Staphylococcus aureus (SAH) was the most promising because it was secreted, caused trauma to cellular membranes, and induced oncosis in 18 min. After exposure for 6 h, SAH decreased cell viability by 90%. In comparison, the positive control, Pseudomonas aeruginosa exotoxin A (PEA), required 11 days to achieve a similar effect, when administered at 3,000 times its LC50 . The maximum death rate induced by SAH was calculated to be a reduction in cell viability of 7.1% per min, which was 200-fold faster than the PEA control. Two proteins, Dermonecrotic Toxin and Phospholipase C were active when extracted from the bacterial cytoplasm but were not secreted. This investigation revealed for the first time SAH as a potent anticancer drug for delivery by bacteria because of its ability to be secreted in a fully functional form and aggressively kill cancer cells.

Lipid A controls the robustness of intratumoral accumulation of attenuated Salmonella in mice.

Engineered Salmonella have the potential to treat cancers that are not responsive to standard molecular therapies. This potential has not been realized because colonization in human tumors is insufficient and variable as shown in preliminary phase I trials. Recent studies have shown that Salmonella colonization is associated with an inflammatory response mediated by tumor necrosis factor (TNF). An injectable agent, molecular lipid A, could be used to control bacterial accumulation because it induces TNF production and is rapidly cleared. We hypothesized that concurrently administrating lipid A with attenuated Salmonella would increase intratumoral accumulation, improve the robustness of tumor-targeting and be nontoxic. To test this hypothesis, Salmonella and lipid A were injected into mice with 4T1 mammary tumors. Colonization was quantified after 48 hr using anti-Salmonella immunofluorescence. A 2 µg/mouse dose of lipid A increased the area of colonized tissue fourfold, reduced variance 50% and ensured colonization in all mice. Comparatively, Salmonella failed to colonize some control mice, similar to human trials. No toxicity was observed in any treated mice. The fraction of tumor tissue with more than 25% bacterial coverage was eight times greater for treated mice compared to controls. Lipid A treatment also reduced the maximum average distance of tissue to Salmonella colonies from 1348 to 260 µm. A mathematical model of bacterial drug production predicted that 2 µg lipid A would increase tumor cell death by 82%. These results suggest that lipid A could solve the clinical challenges of Salmonella therapy and enable safe and robust treatment of cancer with bacteria.

Construction of an inducible cell-communication system that amplifies Salmonella gene expression in tumor tissue.

Bacterial therapies have the potential to overcome resistances that cause chemotherapies to fail. When using bacteria to produce anticancer agents in tumors, triggering gene expression is necessary to prevent systemic toxicity. The use of chemical triggers, however, is hampered by poor delivery of inducing molecules, which reduces the number of activated bacteria. To solve this problem, we created a cell-communication system that enables activated bacteria to induce inactive neighbors. We hypothesized that introducing cell communication into Salmonella would improve direct triggering strategies by increasing protein production, increasing sensitivity to inducer molecules, and enabling expression in tumor tissue. To test these hypotheses we integrated the PBAD promoter into the quorum-sensing machinery from Vibrio fischeri. The expression of a fluorescent reporter gene was compared to expression from non-communicating controls. Function in three-dimensional tissue was tested in a tumor-on-a-chip device. Bacterial communication increased fluorescence 40-fold and increased sensitivity to inducer molecules more than 10,000-fold. The system enabled bacteria to activate neighbors and increased the time-scale of protein production. Gene expression was controllable and tightly regulated. At the optimal inducing signal, communicating bacteria produced 350 times more protein than non-communicating bacteria. The cell-communication system created in this study has uses beyond cancer therapy, including protein manufacturing, bioremediation and biosensing. It would enable amplified induction of gene expression in any environment that limits availability of inducer molecules. Ultimately, because inducible cellular communication enables gene expression in tissue, it will be a critical component of bacterial anticancer therapies.