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Bioconversion of Methanol Into Value- Added Chemicals in Native and Synthetic Methylotrophs

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Bioconversion of Methanol Into Value- Added Chemicals in Native and Synthetic Methylotrophs Bioconversion of Methanol into Value- added Chemicals in Native and Synthetic Methylotrophs Min Zhang1, Xiao-jie Yuan1, Cong Zhang1, Li-ping Zhu1, Xu-hua Mo1, Wen-jing Chen1 and Song Yang1,2* 1School of Life Science, Qingdao Agricultural University, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao, China. 2Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, China. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.033.225 Abstract production of value-added chemicals. In particular, Methanol, commercially generated from methane, methanol is an important one-carbon (C1) feed- is a renewable chemical feedstock that is highly sol- stock that can be generated from either synthesis uble, relatively inexpensive, and easy to handle. Te gas (a mixture of CO and H2) or from biogas, concept of native methylotrophic bacteria serving assuming that large quantities of this feedstock as whole cell catalysts for production of chemicals could be produced at relatively low market price and materials using methanol as a feedstock is (Clomburg et al., 2017; Yang et al., 2018). Methylotrophic highly atractive. In recent years, the available omics bacteria are a diverse group of microbes that can use data for methylotrophic bacteria, especially for reduced C1 compounds such as methanol and Methylobacterium extorquens, the best-characterized methane as sole sources of both energy and carbon model methylotroph, have provided a solid plat- (Chistoserdova et al., 2009; Chistoserdova, 2018). form for rational engineering of methylotrophic Some methylotrophic bacteria, such as Methylo- bacteria for industrial production. In addition, bacterium extorquens, Methylococcus capsulatus, and there is a strong interest in converting the more Methylomicrobium buryatense, show robust growth traditional heterotrophic production platforms under laboratory conditions (Bird et al., 1971; Guo towards the use of single carbon substrates, includ- and Lidstrom, 2006; de la Torre et al., 2015). Te ing methanol, through metabolic engineering. In availability of whole genome sequences, combined this chapter, we review the recent progress towards with biochemical studies (Peyraud et al., 2011; achieving the desired growth and production yields Kalyuzhnaya et al., 2013), have provided important from methanol, by genetically engineered native insights into their metabolism that are crucial for methylotrophic strains and by the engineered syn- enabling engineering these types of microorgan- thetic methylotrophs. ism for bulk chemical production. Meantime, as an alternative approach, more traditional model microorganisms such has Escherichia coli, Introduction Corynebacterium glutamicum and Saccharomyces One goal of metabolic engineering is to develop cerevisiae, have been genetically modifed towards renewable and sustainable alternatives in acquiring the ability to utilize methanol for growth, Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb 226 | Zhang et al. with a potential for converting methanol into value- assimilation (Erb et al., 2007; Peyraud et al., 2009). added compounds (Whitaker et al., 2015; Bennet It has been shown that signifcant metabolic fux et al., 2018b). In this chapter, we review the current goes through the serine cycle and the EMC path- status of the felds of engineering native as well as way when cells are grown on methanol, generating synthetic methylotrophs and provide examples of a stable supply of acetyl-CoA, which could serve successfully engineered strains with a potential in as a precursor for the production of value-added producing commercial value-added chemicals. chemicals in engineered M. extorquens AM1 strains (Fig. 13.1) (Peyraud et al., 2011; Fu et al., 2016). Metabolic engineering of native Production of mevalonate and methylotrophs for value-added terpenoids chemicals production Mevalonate and its derivatives terpenoids are M. extorquens AM1, a pink-pigmented facultative promising bulk chemicals. Terpenoids are used methylotrophic α-proteobacterium capable of as favours, fragrances, pharmaceuticals, and growth on both C1 and multi-carbon compounds, biofuels, representing the largest and the most has served for decades as the model organism for structurally diverse family of chemicals in nature studying C1 metabolism (Chistoserdova et al., (Zhang et al., 2017). To synthesize considerable 2003), including its biotechnological applications amounts of terpenoids, a commonly used strategy (Ochsner et al., 2015). Since the 1960s, it has in bacteria is to introduce an exogenous mevalonate been reported that M. extorquens was successfully (MEV) pathway instead of utilizing the native employed as a producer of certain products such as methylerythritol phosphate (MEP) pathway for amino acids (Sirirote et al., 1988). Based on the cur- circumventing the regulatory control of the MEP rent understanding, M. extorquens employs three pathway (Zhao et al., 2013). Tus, pathway engi- interlocked metabolic cycles for carbon assimi- neering for mevalonate production is a feasible frst lation: the serine cycle, the ethylmalonyl-CoA step as a platform for diverse terpenoid synthesis. (EMC) pathway, and the poly-3-hydroxybutyrate Although E. coli is the primary industrial microbe (PHB) cycle (Fig. 13.1) (Anthony, 2011; Cui et for the production of mevalonate (Nagai et al., al., 2016). Te main function of the EMC pathway 2018), other engineered hosts have the potential to is to regenerate glyoxylate from acetyl-CoA for generate it from alternative carbon feedstocks via reincorporating it into the serine cycle during C1 synthetic metabolic pathways. Zhu and colleagues Figure 13.1 Schematic of methanol metabolism in M. extorquens and target precursors for synthesis of value added compounds. Pink, grey and blue backgrounds represent the primary oxidation module, the dissimilation module, and the assimilation modules, respectively. Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Bioconversion of Methanol in Methylotrophs | 227 engineered a mevalonate pathway by using acetyl- in mevalonate concentration was further achieved CoA as the initial precursor in M. extorquens AM1, following sensor-based high-throughput screening to produce mevalonate from methanol (Zhu et al., of a QscR transcriptional regulator library. Finally, 2016). A natural operon (MVH) harbouring the a fed-batch fermentation in a 5 l bioreactor yielded mvaS and mvaE genes from Enterococcus faecalis, a mevalonate concentration of 2.67 g/l, which was as well as an artifcial operon (MVH) harbouring equivalent to an overall yield of 0.055 mol acetyl- the hmgcs1 gene from Blatella germanica and the CoA/mol methanol. In addition, Cui and colleagues tchmgr gene from Trypanosoma cruzi were con- utilized atmospheric and room temperature plasma structed in a plasmid pCM110. Expression of these (ARTP) mutagenesis, in combination with adap- two operons resulted in the titres of mevalonate of tive laboratory evolution (ALE), generating a 56 mg/l and 66 mg/l, respectively, in fask cultiva- mutant with high methanol tolerance whose cell tions. Further introduction of the phaA gene from density was 7.1-fold higher than that of the parent Ralstonia eutropha into the MVH operon, with the strain in 1.25 M (5%, v/v) methanol (Cui et al., goal of improving the conversion from acetyl-CoA 2018). Accordingly, the mevalonate productivity of into acetoacetyl-CoA increased the mevalonate the mutant strain carrying a mevalonate synthesis titre to 180 mg/l, 3.2-fold higher than that of the pathway was 65% higher than that of the wild-type natural MVE operon. Further modifcation of the strain in methanol fed-batch fermentation. expression level of the phaA gene by regulating In a separate study, Sonntag and colleagues the strength of the ribosomal binding site (RBS) introduced a mevalonate pathway from Myxococcus resulted in an additional increase in mevalonate xanthus, in combination with RBS optimization of production to 215 mg/l (Zhu et al., 2016). Moreo- α-humulene and farnesyl pyrophosphate synthases, ver, it has been demonstrated that, under these resulting in the increase of titres of α-humulene from conditions, the supply of acetyl-CoA was limited 18 mg/l to 54 mg/l in M. extorquens AM1 (Sonn- in engineered M. extorquens AM1. To overcome tag et al., 2015a). Furthermore, the recombinant this limitation, a strategy termed mevalonate M. extorquens AM1 was grown in methanol-limited sensor-assisted transcriptional regulator engineer- fed-batch conditions, with the addition of dode- ing (SATRE) was employed, to control metabolic cane to avoid the accumulation of α-humulene fux redistribution toward increasing acetyl-CoA in the medium. An average OD600 value of 80–90 fux from methanol for mevalonate production were achieved, and fnal product concentrations (Fig. 13.2) (Liang et al., 2017a). A 60% increase were up to 1.65 g/l. Tis is the highest titre of de Figure 13.2 Biosensor-assisted transcriptional regulator engineering approach to increase mevalonate production in M. extorquens AM1. QscR is the regulator of the serine cycle for methanol assimilation (Liang et al., 2017). Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb 228 | Zhang et al. novo synthesized α-humulene reported to date, bioreactor without afecting the biomass yield, with which corresponds to 12% of the maximum theo- the yield of 0.11 g/g from methanol. In addition, retical yield. Te product titres of
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