Synthetic Methanol and Formate Assimilation Via Modular Engineering and Selection Strategies Nico J. Claassens, Hai He and Arren Bar-Even* Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.033.237 Abstract Introduction One-carbon (C1) feedstocks can provide a One-carbon (C1) compounds could prove to be vital link between cheap and sustainable abiotic a crucial link between the abiotic and the biotic resources and microbial bioproduction. Soluble C1 worlds. Tese feedstocks can be obtained from substrates – methanol and formate – could prove low-cost and abundant sources, such as syngas and to be more suitable than gaseous feedstocks as they natural gas (Dürre and Eikmanns, 2015; Clomburg avoid mass transfer barriers. However, microorgan- et al., 2017), and can be produced directly from isms that naturally assimilate methanol and formate CO2 using energy sources such as sunlight and are limited by a narrow product spectrum and a renewable electricity (Kumar et al., 2012; Martín et restricted genetic toolbox. Engineering biotech- al., 2015; Claassens et al., 2018; Jouny et al., 2018). nological organisms to assimilate these soluble C1 Multiple microorganisms can be cultivated on C1 substrates has therefore become an atractive goal. compounds as sole carbon and energy sources, thus Here, we discuss the use of a step-wise, modular opening new avenues for sustainable bioproduc- engineering approach for the implementation of tion. C1 assimilation pathways. In this strategy, pathways However, the use of microorganisms that can are divided into metabolic modules, the activities naturally grow on C1 substrates is limited by mul- of which are selected for in dedicated gene-deletion tiple factors, including a narrow product spectrum, strains whose growth directly depends on module low yields, titres, and productivities, a restricted activity. Tis provides an easy way to identify and genetic toolbox for engineering, and gaps in our resolve metabolic barriers hampering pathway per- understanding of their cellular physiology and formance. Optimization of gene expression levels metabolism (Whitaker et al., 2015; Clomburg et al., and adaptive laboratory evolution can be used to 2017). To overcome these difculties, recent meta- establish the desired activity if direct selection fails. bolic engineering eforts are aiming to introduce We exemplify this approach using several pathways, C1 assimilation pathways into model biotechno- focusing especially on the ribulose monophosphate logical microorganisms that are easier to engineer cycle for methanol assimilation and the reduc- and that can be beter optimized for industrially tive glycine pathway for formate assimilation. We relevant conditions. Tese eforts use either natural argue that such modular engineering and selection pathways that are known to sustain high yields, or, strategies will prove essential for rewiring microbial more boldly, synthetic pathways with low ATP cost metabolism towards new growth phenotypes and that could theoretically support increased yields sustainable bioproduction. (Bar-Even et al., 2013; Siegel et al., 2015; Bar-Even, Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb 238 | Claassens et al. 2016;). Some of these synthetic pathways can Modularity and selection be established by combining naturally existing as metabolic engineering enzymes, while others include novel enzyme activi- strategies ties that can be realized by protein engineering Engineering synthetic C1 metabolism requires (Erb et al., 2017). In fact, engineered enzymes have the overexpression of pathway enzymes, especially already been demonstrated in vitro to support for- those that are missing in the host or that are natively mate assimilation (Siegel et al., 2015) and carbon expressed at insufcient levels. However, simple fxation (Schwander et al., 2016). overexpression is unlikely to be sufcient for realiz- In this review, we discuss metabolic engineer- ing the activity of the entire pathway. Tis is mainly ing studies aiming to introduce pathways for the because of the overlap between the introduced assimilation of the soluble C1 compounds metha- pathway and the host central metabolism, result- nol and formate, the utilization of which bypasses ing in disrupted fuxes through both systems. To the challenges associated with mass transfer of beter identify and resolve problematic metabolic gaseous C1 substrates, such as methane and carbon interactions, it is helpful to divide the synthetic monoxide (Henstra et al., 2007; Fei et al., 2014). pathway into smaller metabolic modules, i.e. sub- We specifcally focus on modular and selection- pathways consisting of several reactions (Fig. 14.1). based engineering strategies in which the activity Te in vivo implementation of these modules can of pathway segments is coupled to cellular growth. be considerably easier than the full pathway and We show that this step-wise approach is vital for the provide vital information on the metabolic context realization of synthetic C1 assimilation. that enables or constrains the newly introduced 1 2 3 4 Divide synthetic pathway Multiple expression levels for into several metabolic each enzyme are tested, modules, each corresponds for example by varying to a discrete metabolic goal strength of promoters, RBS, and plasmid origins of replication Express each module in a low high dedicated auxotrophic selection selection strain, the growth of which Increase selection for module strictly depends upon Fraction of cells activity in different strains, the module activity, enabling growth of each requires a direct selection for activity Protein level (log scale) different level of module activity If direct selection fails, or Feeding with 13C-labeled formate results in sub-optimal growth, (or another carbon source) and + long-term cultivation under monitoring the labeling pattern in selective conditions proteinogenic amino acids, we (chemostat or turbidostat) can confirm module activity or = evolves desired growth identify competing routes that properties should be deleted Sequence evolved strains, Integrate modules into a full O O feed introduce mutations to a H pathway within a strain carrying naïve strain to identify the 2 the beneficial mutations previously HC C H C contribution of each mutation, OH HO C OH identified, and select for growth via identify minimal set of the pathway, upon expression of the effluent mutations to enable activity NH2 (potentially evolved) pathway enzymes 5 6 7 8 Figure 14.1 A schematic representation of the modular engineering and selection approach outlined in this paper. Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Strategies for Synthetic C1 Assimilation | 239 activities. To probe the implementation of meta- and potentially also of related host enzymes, e.g. bolic modules, it is useful to couple their activity deletion or down-regulation of enzymes that with the growth of the host. divert metabolic intermediates from the pathway. Coupling module activity with growth usu- In addition, diferent enzyme variants or codon ally requires modifying the metabolic network of optimization of the relevant genes can sup- the host by performing strategic gene deletions. port increased expression and activity. Another Tese are made to generate a strain auxotrophic method, which does not rely on genetic tools, is for certain essential metabolic intermediates – for the addition of small molecules that specifcally example, an amino acid – which can be exclusively inhibit interfering enzymes, as demonstrated synthesized via the synthetic module. As a result, for the glycolytic glyceraldehyde 3-phosphate cellular growth becomes dependent on the activ- dehydrogenase in the engineering of methanol ity of the module. A range of selection strains can assimilation in Escherichia coli (Woolston et al., be designed with increasing selection pressure for 2018a). pathway activity: a ‘minimal’ selection is sustained If these approaches fail to establish module if the module provides a single required metabo- activity, adaptive laboratory evolution (ALE) can lite, higher selection pressure is obtained when be performed to increase module functionality module activity is responsible for the biosynthesis and establish module-dependent growth (Portnoy of multiple building blocks, and very high selection et al., 2011). For this process, the overexpressed pressure is imposed when the biosynthesis of all or genes should preferably be integrated into the most biomass is dependent on the module. genome rather than carried on a plasmid as to Te design of modules and selection strains increase the chance of benefcial mutations to be can be assisted by computational tools based on fxed in the population. Diferent types of ALE Flux Balance Analysis, for example OptKnock can be applied; a prominent approach being con- or FlexFlux (Burgard et al., 2003; Marmiesse et tinuous cultivation on a selective medium, with al., 2015; Meyer et al., 2018). Yet, in most cases, limiting amounts of the compounds for which the manual design based on biochemical and metabolic cells are auxotrophic. Tis method was applied knowledge sufces. Specifcally, when dividing a for the successful engineering of the CO2-fxing pathway into metabolic modules, several factors Calvin cycle in E. coli (Antonovsky et al., 2016), should be taken into consideration (Wenk et al., where the concentration of the limiting sub- 2018): (1) the module should be linked