Recombinant Host Cell FLO8 Knockout Glucose-Regulated Protein Production
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Solution Overview
Problem
Current methods for producing recombinant proteins in host cells face challenges in optimizing protein yield and reducing the risk of host cell morphology changes during production, particularly due to the expression of FLO8 proteins which can hinder protein production efficiency.
Innovation Solution
Engineering a recombinant host cell to reduce the expression of FLO8 proteins by genetic modifications, such as knockout or disruption of the FLO8 gene, and using a heterologous expression cassette with a promoter that is regulatable by a non-methanol carbon source like glucose or glycerol, allowing for controlled protein production without methanol induction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If FLO8 protein expression is maintained in host cells, then host cell morphology is preserved, but protein production yield is reduced
Solution Approach 1:
The FLO8 gene is knocked out or disrupted in advance before protein production begins, preventing the formation of harmful flocculins that would otherwise reduce protein yield. This preliminary genetic modification eliminates the conflict between maintaining host cell morphology and achieving high productivity, as the harmful FLO8 protein is never expressed during the production process
2Productivity
If methanol-controlled promoters are used for protein expression, then protein production can be induced, but technical limitations such as waste heat and oxygen supply issues occur
Solution Approach 1:
The patent replaces methanol-controlled promoters with carbon-source regulatable promoters (such as glucose-regulated promoters) that respond to different chemical signals. This parameter change in promoter regulation allows protein expression to be controlled through carbon source availability rather than methanol induction, thereby avoiding the waste heat and oxygen supply problems associated with methanol metabolism while maintaining inducible protein production capability
Data Source
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AI summary
A recombinant host cell comprising an endogenous gene encoding a FLO8 protein comprising the amino acid sequence identified as SEQ ID NO:1 or a homologue thereof, which host cell is engineered by one or more genetic modifications to reduce expression of said gene compared to the host cell prior to said one or more genetic modifications, and which host cell comprises a heterologous expression cassette comprising a gene of interest (GO!) under the control of an expression cassette promoter (ECP) which ECP is repressible by a non-methanol carbon source, and a method of producing a protein of interest using said recombinant host cell.