Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway

Abstract

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

Breeding of Methanol-Tolerant Methylobacterium extorquens AM1 by Atmospheric and Room Temperature Plasma Mutagenesis Combined With Adaptive Laboratory Evolution

“…It was cultivated in LB medium (1% tryptone, 0.5% yeast extract, and 0.5% sodium chloride) containing appropriate antibiotics. In M. extorquens AM1 purchased from NCIMB(NCIMB 9133; Aberdeen, Scotland), cel gene was knocked out, and the resulting strain was named as AM1‐dcel . Small‐scale cultivation was performed in MC medium as described, with addition of antibiotics as needed .…”

“…Methanol is a globally distributed C1 product that is an ideal alternative carbon feedstock for industrial biotechnology . Methylotrophic cell factories (MeCFs), which are based on methanol‐utilizing bacteria, are a promising platform to produce chemicals and biofuels from methanol …”

“…These attributes make M. extorquens AM1 an ideal platform on which to develop methanol‐based biomanufacturing. As a result of metabolic engineering, M. extorquens AM1 has been used to produce polyhydroxyalkanoates (PHAs), mevalonate acid (MEV), α‐humulene, and 1‐butanol . However, higher concentrations of methanol and sudden shifts in its concentration have toxic effects on these bacteria .…”

“…The possible function of mutant genes were also analysis by gene overexpression in parent strain. Finally, to test the applicability of this high‐methanol‐tolerant strain, the mvt operon for the synthesis of MEV was introduced into the mutant. The improved fermentation performance of the recombinant mutant was examined by flask culture with medium containing 5% methanol‐contained medium in terms of cell growth and MEV production.…”

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

“…A well-studied representative is Methylobacterium extorquens AM1, a pink-pigmented, aerobic alphaproteobacterium first identified in 1961 (18). This model methylotroph has already been applied for the synthesis of industry-relevant products, for example, mevalonic acid, mesaconic acid, and methylsuccinic acid, from methanol (19,20). The exceptional metabolism of this bacterium was not completely elucidated until recently (21,22).…”