Designing and Engineering Methylorubrum extorquens AM1 for Itaconic Acid Production

Abstract

Methylorubrum extorquens (formerly Methylobacterium extorquens ) AM1 is a methylotrophic bacterium with a versatile lifestyle. Various carbon sources including acetate, succinate and methanol are utilized by M. extorquens AM1 with the latter being a promising inexpensive substrate for use in the biotechnology industry. Itaconic acid (ITA) is a high-value building block widely used in various industries. Given that no wildtype methylotrophic bacteria are able to utilize methanol to produce ITA, we tested the potential of M. extorquens AM1 as an engineered host for this purpose. In this study, we successfully engineered M. extorquens AM1 to express a heterologous codon-optimized gene encoding cis- aconitic acid decarboxylase. The engineered strain produced ITA using acetate, succinate and methanol as the carbon feedstock. The highest ITA titer in batch culture with methanol as the carbon source was 31.6 ± 5.5 mg/L, while the titer and productivity were 5.4 ± 0.2 mg/L and 0.056 ± 0.002 mg/L/h, respectively, in a scaled-up fed-batch bioreactor under 60% dissolved oxygen saturation. We attempted to enhance the carbon flux toward ITA production by impeding poly-β-hydroxybutyrate accumulation, which is used as carbon and energy storage, via mutation of the regulator gene phaR . Unexpectedly, ITA production by the phaR mutant strain was not higher even though poly-β-hydroxybutyrate concentration was lower. Genome-wide transcriptomic analysis revealed that phaR mutation in the ITA-producing strain led to complex rewiring of gene transcription, which might result in a reduced carbon flux toward ITA production. Besides poly-β-hydroxybutyrate metabolism, we found evidence that PhaR might regulate the transcription of many other genes including those encoding other regulatory proteins, methanol dehydrogenases, formate dehydrogenases, malate:quinone oxidoreductase, and those synthesizing pyrroloquinoline quinone and thiamine co-factors. Overall, M. extorquens AM1 was successfully engineered to produce ITA using acetate, succinate and methanol as feedstock, further supporting this bacterium as a feasible host for use in the biotechnology industry. This study showed that PhaR could have a broader regulatory role than previously anticipated, and increased our knowledge of this regulator and its influence on the physiology of M. extorquens AM1.

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

“…When cultivated on media containing formate or methanol, the metabolic flux largely goes through the tetrahydrofolate, serine, and EMC pathways, generating a stable supply of acetyl-CoA, which could serve as a precursor for the production of value-added chemicals in engineered M. extorquens AM1 strains ( Peyraud et al, 2011 ; Zhang et al, 2019 ; Hwang et al, 2020 ). This strain has been engineered to produce a variety of value-added chemicals or biofuels such as PHB ( Hwang et al, 2020 ; Yoon et al, 2021 ), polyhydroxyalkanoate terpolymers ( Orita et al, 2014 ), 1-butanol ( Hu and Lidstrom, 2014 ), 3-hydroxypropionic acid (3-HP) ( Yang et al, 2017 ), mevalonic acid ( Zhu et al, 2016 ), butadiene precursor ( Yang et al, 2018 ), terpenoids ( Sonntag et al, 2015a ), mesaconic acids ( Sonntag et al, 2015b ), crotonic acids ( Schada Von Borzyskowski et al, 2018 ), and itaconic acids ( Lim et al, 2019 ) from formate or methanol.…”

In silico predictions of protein interactions between Zika virus and human host

“…We could see some expected enriched pathways normally modulated in viral infections such as apoptosis, cell cycle, and endocytosis, however, some more peculiar pathways were also identified such as the axon guidance pathway. The main cell controlling the axonal guidance is the astrocyte, and it is one of the targets of ZIKV infection ( Limonta et al, 2018 ; Sher, Glover & Coombs, 2019 ). This could be involved directly in microcephaly symptoms, and we could see several proteins from the host interacting with ZIKV proteins ( Limonta et al, 2018 ).…”

Chemical Production from Methanol Using Natural and Synthetic Methylotrophs

“…Engineered M. extorquens strain expressing a cis ‐aconitate decarboxylase (Cad) from ( A. terreus ) resulted in the production of itaconic acid up with a small titer of 31.6 mg L −1 from methanol. [ 23 ] This titer is significantly lower than that of itaconic acids produced from sugar using engineered E. coli , [ 65 ] which has been extensively studied. Although it is difficult to pin‐point the exact bottleneck for methanol‐based itaconic acid production, metabolic engineering approaches that have shown to increase TCA cycle dependent products should be applied to M. extorquens AM1 for improving itaconic acid production.…”