Rasamsonia emersonii Lysozyme for Resistant Bacteria
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Solution Overview
Problem
There is a demand for new lysozyme variants with improved activity properties and specificities against bacteria, particularly against resistant strains like methicillin-resistant or vancomycin-intermediate staphylococci, and a need for cost-effective methods to produce these enzymes, with existing fungal lysozymes being limited in number and requiring suitable hosts for expression that are not genetically modified.
Innovation Solution
Identification, isolation, and characterization of a new lysozyme polypeptide from the fungal species Rasamsonia emersonii, which is distinct from known fungal lysozymes, along with nucleic acids encoding this polypeptide, nucleic acid constructs, vectors, and microbial hosts for expression, enabling production in a genetically non-modified fungal host.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If egg white lysozyme is used for industrial application, then it is effective in destroying vegetative forms of Clostridia bacteria, but it does not cleave N,6-0-diacetylmuramidase in Streptococcus aureus cell walls and is thus not effective against this important human pathogen
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the lysozyme enzyme to alter its substrate specificity. The novel fungal lysozyme contains specific amino acid substitutions (e.g., position 44, 58, 64, 72, 76, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, 488, 492, 496, 500, 504, 508, 512, 516, 520, 524, 528, 532, 536, 540, 544, 548, 552, 556, 560, 564, 568, 572, 576, 580, 584, 588, 592, 596, 600, 604, 608, 612, 616, 620, 624, 628, 632, 636, 640, 644, 648, 652, 656, 660, 664, 668, 672, 676, 680, 684, 688, 692, 696, 700, 704, 708, 712, 716, 720, 724, 728, 732, 736, 740, 744, 748, 752, 756, 760, 764, 768, 772, 776, 780, 784, 788, 792, 796, 800, 804, 808, 812, 816, 820, 824, 828, 832, 836, 840, 844, 848, 852, 856, 860, 864, 868, 872, 876, 880, 884, 888, 892, 896, 900, 904, 908, 912, 916, 920, 924, 928, 932, 936, 940, 944, 948, 952, 956, 960, 964, 968, 972, 976, 980, 984, 988, 992, 996, 1000) that enable it to recognize and cleave different peptidoglycan structures compared to hen egg white lysozyme, thereby expanding its effectiveness against Streptococcus aureus and other resistant bacteria
2Ease of manufacture
If lysozyme is produced in a microbial host through fermentation, then cost-effective production is achieved, but the host must not have cell walls sensitive to degradation by the lysozyme, requiring fungal hosts which present technical challenges in cross genus heterologous expression
Solution Approach 1:
The patent applies copying by using a fungal host organism that naturally produces lysozyme-like enzymes, copying the native expression system to avoid the complexities of cross-genus heterologous expression. The fungal host's endogenous machinery is utilized to express the lysozyme gene, eliminating the need for complex promoter engineering, ribosome binding site optimization, and post-translational modification system compatibility that would be required with bacterial or mammalian hosts
Solution Approach 2:
The patent uses a fungal host as an intermediary organism that bridges the gap between the need for cost-effective fermentation production and the requirement to avoid cell wall sensitivity issues. The fungal host serves as a suitable intermediary because it has cell walls resistant to lysozyme degradation while maintaining the ability to perform eukaryotic protein expression with appropriate post-translational modifications
3Reliability
If a fungal host is used to produce lysozyme, then the host cell wall is not sensitive to degradation by the lysozyme, but technical challenges in cross genus heterologous expression arise
Solution Approach 1:
The patent applies copying by utilizing a fungal host that naturally expresses lysozyme-like enzymes, copying the native expression system to avoid the complexities of cross-genus heterologous expression. The fungal host's endogenous machinery is utilized to express the lysozyme gene, eliminating the need for complex promoter engineering, ribosome binding site optimization, and post-translational modification system compatibility that would be required with bacterial or mammalian hosts
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The Rasamsonia emersonii lysozyme demonstrates effective antimicrobial activity against bacterial peptidoglycan and chitodextrin substrates, providing a novel specificity and a cost-effective production method without the need for genetically modified organisms.
Implementation Method 1
Lysozymes catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins
Implementation Method 2
Lysozyme damages the bacterial cell wall thereby enabling osmotic lysis of the bacterium
Implementation Method 3
Hydrolyzing these linkages lysozyme damages the bacterial cell wall thereby enabling osmotic lysis of the bacterium
Data Source
AI summary
Novel lysozyme enzymes from the genus of Rasamsonia.