Recombinant Pectate Lyase Expression for Industrial Pectin Breakdown
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current technologies lack efficient methods for cloning and utilizing pectin degrading enzymes from M. phaseolina, which are crucial for industrial processes such as fruit and vegetable juice extraction, textile processing, and paper production, due to the unavailability of genetic information on these enzymes from this specific fungus.
Innovation Solution
The disclosure provides nucleotide sequences encoding pectate lyases from M. phaseolina, along with expression vectors and host cells, enabling the in vitro production of pectin degrading polypeptides, facilitating their use in various industrial applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If pectin degrading enzymes are produced by traditional methods using other fungal sources, then commercial production is possible, but the specific industrial applications requiring M. phaseolina enzymes cannot be met
Solution Approach 1:
The patent creates copies of the pectate lyase gene from M. phaseolina and inserts it into expression vectors, which are then transformed into host cells to produce recombinant enzymes. This copying approach allows the specific genetic information to be replicated and utilized for industrial applications without requiring large quantities of the original fungal material.
Solution Approach 2:
The patent uses expression vectors and host cells as intermediaries to bridge the gap between the unavailable genetic information and the desired enzyme production. The vector system acts as a mediator that carries the genetic information into suitable host organisms that can then produce the enzymes for industrial use.
2Ease of manufacture
If genetic information on pectate lyases from M. phaseolina is made available, then in vitro production becomes possible, but the complexity of cloning and expression systems increases
Solution Approach 1:
The patent employs universal expression vectors and host cell systems that can be used for producing multiple different enzymes, not just pectate lyases. This multi-functionality reduces the overall complexity by using standardized, well-characterized systems rather than developing specialized complex systems for each enzyme production.
Solution Approach 2:
The patent uses transient expression systems and disposable expression vectors that can be easily introduced into host cells for short-term enzyme production. These systems are designed to be simple, inexpensive, and easily replaceable, reducing the complexity of maintaining long-term complex genetic systems.
3Productivity
If recombinant expression systems are used for enzyme production, then large scale commercial production is achievable, but the process requires multiple steps including vector construction, transformation, and purification
Solution Approach 1:
The patent performs preliminary optimization of the expression vectors and host cell systems before large-scale production. Expression conditions, purification tags, and vector elements are pre-optimized to ensure high productivity, reducing the need for multiple iterative steps during actual production and streamlining the overall process.
Solution Approach 2:
The patent combines multiple functions into integrated expression systems where gene expression, protein folding, and initial purification steps are merged into a single transformation and cultivation process. This reduces the number of separate operational steps required for commercial production while maintaining high productivity.
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
This approach allows for the commercial production of pectin degrading enzymes from M. phaseolina, meeting the global demand for products like fruit juice, textiles, pulp, and paper, while enhancing efficiency in coffee and tea fermentation and oil extraction.
Implementation Method 1
Pectin/pectate lyases depolymerize pectin in smooth region, which cleaves glycosidic bonds via beta-elimination to yield oligomers that are 4, 5-unsaturated non-reducing end
Implementation Method 2
Pectin esterase hydrolyses the pectin to methanol and polygalacturonic acid
Data Source
Figure 1~3
Figure 4~6
Figure 7~9
AI summary
The present invention discloses isolated polynucleotide encoding enzymes, derived from the fungus Macrophomina phaseolina ("M. phaseolina"), responsible for degrading pectin, and it comprises and/or consists of nucleotide sequences set forth in SEQ. ID Nos. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61, or the complement of such sequences. The present invention also relates to isolated polypeptide encoded by the polynucleotide sequences set forth in SEQ ID Nos. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 and 63; a recombinant gene construct comprising the polynucleotide; a transformant and a transgenic fungus comprising the recombinant gene construct, with or having enhanced production of pectin degrading enzyme. The polypeptide of the invention can be used for, amongst other things, manufactured fruit juice, textile products, pulp and paper, coffee, tea and oil extraction and pectic waste water treatment.