SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 3, 2019, is named GTRC7595_011529_113444_SL.txt and is 57,460 bytes in size.

BACKGROUND OF THE DISCLOSURE

1. Field of the Disclosure

Embodiments of the present disclosure relate generally to modifying amino acids at the interface between interacting enzymes in order to alter their binding, and more specifically to engineering the interacting surface of heterologous thioesterase (TE) enzymes to improve binding to endogenous host fatty acyl-acyl carrier proteins (fatty acyl-ACPs).

2. Background

The introduction of heterologous proteins into a host cell faces numerous hurdles, from proper expression of the protein to proper folding to proper function in the host cell. When introducing a heterologous protein that is intended to interact with an endogenous host protein, amino acids at the surface of the heterologous protein may need to be substituted in order to improve the interaction while maintaining the protein's function.

Heterologous proteins are often introduced into bacterial cells to express molecules of interest. Many times, these molecules are either not produced at all by the bacteria or are produced in low amounts. For example, microbial production of medium-chain fatty acids (MCFAs; includes C8-C12 backbones) is limited by the activity and product profile of bacterial enzymes, specifically bacterial acyl-ACP thioesterases. MCFAs are useful as antimicrobials and emulsifying agents and can be derivatized to form a number of useful chemical intermediates (e.g., alkenes, α-olefins, esters, alcohols, ketones, hydroxyacids such as w-hydroxy-carboxylic acids, and dicarboxylic acids such as α,α-dicarboxylic acids).

Previous attempts to synthesize MCFAs in bacteria included providing plant thioesterases (TEs) that were not efficiently expressed in the host as well as mutagenizing amino acids near the active site of bacterial TEs in an attempt to change the fatty acids produced by the enzymes to MCFAs. For example, the TE that interacts with AcpP inE. coli, TesA, has frequently been mutagenized near its active site in order to improve MCFA production without much success.

What is needed, therefore, is a method of engineering a heterologous thioesterase enzyme to interact with endogenous acyl-ACP proteins in order to produce molecules of interest in a bacterial host. The method should identify amino acids at the binding interface between the heterologous TE and the endogenous acyl-ACP that can be substituted so that the heterologous TE can better interact with the endogenous acyl-ACP. Mutant heterologous TE enzymes with improved interactions with the endogenous acyl-ACP are also provided. It is to such a method that embodiments of the present disclosure are directed.

BRIEF SUMMARY OF THE DISCLOSURE

As specified in the Background Section, there is a great need in the art to identify technologies for protein engineering and use this understanding to develop novel methods of engineering heterologous enzymes to interact with endogenous proteins in order to produce molecules of interest in a bacterial host. The present disclosure satisfies this and other needs. Embodiments of the present disclosure relate generally to modifying amino acids at the interface between interacting enzymes in order to alter their binding, and more specifically to engineering the interacting surface of heterologous thioesterase enzymes to improve binding to endogenous host acyl-ACP proteins.

In one aspect, the disclosure provides a method for generating a thioesterase mutant comprising: selecting a heterologous thioesterase for mutation based at least in part on a desired end product that is produced by the heterologous thioesterase; identifying amino acids on the heterologous thioesterase that form an interacting surface withE. coliAcpP and are suitable for mutation; mutagenizing the identified amino acids of the heterologous thioesterase by introducing substitutions into a nucleic acid comprising the heterologous thioesterase nucleotide sequence; and expressing the nucleic acid comprising the mutated heterologous thioesterase nucleotide sequence in a bacterial cell.

In another aspect, the disclosure provides a heterologous thioesterase mutant produced by a method comprising: identifying a heterologous thioesterase for mutation based at least in part on a desired end product that is produced by the heterologous thioesterase; identifying amino acids in the heterologous thioesterase amino acid sequence that form an interacting surface with anE. coliAcpP protein and are suitable for mutation; mutagenizing the identified amino acids of the heterologous thioesterase by introducing substitutions into a nucleic acid comprising the heterologous thioesterase nucleotide sequence; and expressing the nucleic acid comprising the mutated heterologous thioesterase nucleotide sequence in a bacterial cell.

These and other objects, features and advantages of the present disclosure will become more apparent upon reading the following specification in conjunction with the accompanying description, claims and drawings.

