Compositions and methods for production of myrcene

Inventors

Main, Andrew • Wojciechowski, Grzegorz • Yang, Yue • Zhao, Lishan

Assignees

Amyris Inc

Abstract

Provided herein are compositions and methods for producing myrcene by culturing genetically modified microbial host cells that express a myrcene synthase and optionally a geranyl pyroplosphate synthase. Also provided herein are isolated nucleic acid molecules that encode myrcene synthase variants derived from the Ocimum species myrcene synthase, which comprise one or more amino acid substitutions that improve in vivo performance of myrcene synthase in genetically modified microbial host cells. Also provided herein are isolated myrcene synthase variants that exhibit an improved activity for converting geranyl diphosphate into myrcene.

Core Innovation

The invention relates to genetically modified microbial host cells and fermentation compositions used to produce myrcene. The host cells comprise heterologous nucleic acids encoding an Ocimum species myrcene synthase and a geranyl pyrophosphate synthase, where the myrcene synthase has at least 70% sequence identity to SEQ ID NO: 2 and at least one variant amino acid residue at selected positions numbered with reference to SEQ ID NO: 2.

A further aspect includes adding heterologous mevalonate pathway genes and functionally disrupting endogenous farnesyl pyrophosphate synthase to redirect carbon flow to geranyl pyrophosphate, and describes strategies to minimize metabolic cross talk by using the DXP versus MEV IPP pathways, including functional disabling of the DXP pathway and exclusive reliance on the MEV IPP pathway. The document also describes modifying pathway enzymes by sequence variants, codon optimization, directed evolution, rational mutagenesis, feedback or feed-forward regulation changes, and homolog discovery and use of homologous enzymes.

The document further describes myrcene-producing systems in genetically modified microbes expressing myrcene biosynthesis enzymes and GPPS, with culture medium and suitable culture conditions for producing myrcene. Example results described include Ocimum basilicum myrcene synthase variants showing improved relative activity and higher myrcene production in yeast, and product profiling to evaluate myrcene purity and fingerprints.

Claims Coverage

The document includes two independent claims. The coverage centers on an Ocimum-derived myrcene synthase sequence defined by identity to SEQ ID NO: 2 plus variant residue positions, combined with a geranyl pyrophosphate synthase in a genetically modified microbial host cell, and on an isolated nucleic acid molecule encoding the corresponding myrcene synthase.

Overall, the independent-claim coverage is directed to specified Ocimum-derived myrcene synthase proteins defined by SEQ ID NO: 2 identity plus variant residue positions and, for the host cell, to co-expression via heterologous nucleic acids that include a geranyl pyrophosphate synthase. The nucleic-acid independent claim narrows to isolated nucleic acids encoding the corresponding myrcene synthase with the same identity and variant-position constraints.

Stated Advantages

Documented Applications