Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

/pdf/fuelling-the-future-microbial-engineering-for-the-production-4zayx8tgii.pdf

Fuelling the future: microbial engineering for the production of sustainable biofuels

The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where al...

Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules

Producing mass quantities of chemicals has its roots in the industrial revolution. But industrial synthesis leads to sizeable sustainability and socioeconomic challenges. The rapid advances in biotechnology suggest that biological manufacturing may soon be a feasible alternative, but can it produce chemicals at scale? Clomburg et al. review the progress made in industrial biomanufacturing, including the tradeoffs between highly tunable biocatalysts and units of scale. The biological conversion of single-carbon compounds such as methane, for example, has served as a testbed for more sustainable, decentralized production of desirable compounds.

Science , this issue p. [10.1126/science.aag0804][1]

 [1]: /lookup/doi/10.1126/science.aag0804

http://science.sciencemag.org/content/sci/355/6320/aag0804.full.pdf

Industrial biomanufacturing: The future of chemical production

The increasing availability of renewable electricity at costs competitive with, and even lower than, electricity from fossil sources along with growing interest and recent technological advancements in reducing carbon emissions through CO2 capture is challenging the status quo in the way that we produce and consume energy and products. Renewable electricity can be leveraged to produce fuels and chemicals from CO2, offering sustainable routes to reduce the carbon intensity of our energy and products-driven economy. A number of approaches have been developed for the electron-driven reduction of CO2 to products, including both direct and indirect (via an energy carrier such as H2) pathways and spanning from electrochemical to biological to thermocatalytic conversion. While these approaches are at various stages of development, there are technical barriers related to each core conversion technology that need to be addressed in order to accelerate commercialization and drive the transition towards a circular carbon economy. In this perspective, we assess and characterize the top technical barriers for utilizing renewable electricity for CO2 reduction across five different conversion approaches (direct electrochemical, direct bioelectrochemical, direct non-thermal plasma, indirect bioelectrochemical, and indirect thermochemical) under state-of-technology conditions, outline the R&D needs to overcome each barrier, and identify the most promising C1–C3 hydrocarbons and oxygenates based on their relative ease of formation, economic viability, CO2 utilization potential, and energy storage capacity. Our analysis suggests, based on current reported states of technology, that indirect pathways paired with the formation of C1 products offer the most technically feasible approach for electron driven CO2 reduction in the near term. However, as we strive for longer carbon chain molecules, and as technologies continue to advance, there are a multitude of advantages and limitations to be considered for all five approaches.

Transforming the carbon economy: challenges and opportunities in the convergence of low-cost electricity and reductive CO2 utilization

https://escholarship.org/content/qt5mz1s1pz/qt5mz1s1pz.pdf?t=p9sqjd

Escherichia coli as a host for metabolic engineering.

Methanol is an important intermediate in the utilization of natural gas for synthesizing other feedstock chemicals. Typically, chemical approaches for building C–C bonds from methanol require high temperature and pressure. Biological conversion of methanol to longer carbon chain compounds is feasible; however, the natural biological pathways for methanol utilization involve carbon dioxide loss or ATP expenditure. Here we demonstrated a biocatalytic pathway, termed the methanol condensation cycle (MCC), by combining the nonoxidative glycolysis with the ribulose monophosphate pathway to convert methanol to higher-chain alcohols or other acetyl-CoA derivatives using enzymatic reactions in a carbon-conserved and ATP-independent system. We investigated the robustness of MCC and identified operational regions. We confirmed that the pathway forms a catalytic cycle through 13C-carbon labeling. With a cell-free system, we demonstrated the conversion of methanol to ethanol or n-butanol. The high carbon efficiency and low operating temperature are attractive for transforming natural gas-derived methanol to longer-chain liquid fuels and other chemical derivatives.

https://www.pnas.org/content/pnas/111/45/15928.full.pdf

Building carbon–carbon bonds using a biocatalytic methanol condensation cycle

The heavy dependence on petroleum-derived fuel has raised concerns about energy sustainability and climate change, which have prompted researchers to explore fuel production from renewable sources. 1-Butanol and isobutanol are promising biofuels that have favorable properties and can also serve as solvents or chemical feedstocks. Microbial production of these alcohols provides great opportunities to access a wide spectrum of renewable resources. In recent years, research has improved the native 1-butanol production and has engineered isobutanol production in various organisms to explore metabolic diversity and a broad range of substrates. This review focuses on progress in metabolic engineering for the production of these two compounds using various resources.

Frontiers in microbial 1-butanol and isobutanol production

Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production.

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

/pdf/characterization-and-evolution-of-an-activator-independent-4ni7f8h81t.pdf

Chang-Ting Chen

Papers

Building carbon–carbon bonds using a biocatalytic methanol condensation cycle

Frontiers in microbial 1-butanol and isobutanol production

Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production.

Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

Corrigendum to "Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production" [Metab. Eng. 49 (2018) 257-266].