Parageobacillus thermoglucosidasius as an emerging thermophilic cell factory.

Parageobacillus thermoglucosidasius is a thermophilic and facultatively anaerobic microbe, which is emerging as one of the most promising thermophilic model organisms for metabolic engineering. The use of thermophilic microorganisms for industrial bioprocesses provides the advantages of increased reaction rates and reduced cooling costs for bioreactors compared to their mesophilic counterparts. Moreover, it enables starch or lignocellulose degradation and fermentation to occur at the same temperature in a Simultaneous Saccharification and Fermentation (SSF) or Consolidated Bioprocessing (CBP) approach. Its natural hemicellulolytic capabilities and its ability to convert CO to metabolic energy make P. thermoglucosidasius a potentially attractive host for bio-based processes. It can effectively degrade hemicellulose due to a number of hydrolytic enzymes, carbohydrate transporters, and regulatory elements coded from a genomic cluster named Hemicellulose Utilization (HUS) locus. The growing availability of effective genetic engineering tools in P. thermoglucosidasius further starts to open up its potential as a versatile thermophilic cell factory. A number of strain engineering examples showcasing the potential of P. thermoglucosidasius as a microbial chassis for the production of bulk and fine chemicals are presented along with current research bottlenecks. Ultimately, this review provides a holistic overview of the distinct metabolic characteristics of P. thermoglucosidasius and discusses research focused on expanding the native metabolic boundaries for the development of industrially relevant strains.

Engineering the biological conversion of formate into crotonate in Cupriavidus necator.

To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO2 and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacterium C. necator as cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growing C. necator in minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacterium via a modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse ß-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 ± 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical.

The cofactor challenge in synthetic methylotrophy: bioengineering and industrial applications.

Methanol is a promising feedstock for industrial bioproduction: it can be produced renewably and has high solubility and limited microbial toxicity. One of the key challenges for its bio-industrial application is the first enzymatic oxidation step to formaldehyde. This reaction is catalysed by methanol dehydrogenases (MDH) that can use NAD+, O2 or pyrroloquinoline quinone (PQQ) as an electron acceptor. While NAD-dependent MDH are simple to express and have the highest energetic efficiency, they exhibit mediocre kinetics and poor thermodynamics at ambient temperatures. O2-dependent methanol oxidases require high oxygen concentrations, do not conserve energy and thus produce excessive heat as well as toxic H2O2. PQQ-dependent MDH provide a good compromise between energy efficiency and good kinetics that support fast growth rates without any drawbacks for process engineering. Therefore, we argue that this enzyme class represents a promising solution for industry and outline engineering strategies for the implementation of these complex systems in heterologous hosts.

A Hitchhiker's Guide to Supplying Enzymatic Reducing Power into Synthetic Cells.

The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-of-equilibrium thermodynamic status hallmarking life, including the essential supply of energy in the form of electrons. The nicotinamide cofactors NAD(H) and NADP(H) are the main electron carriers fueling reductive redox reactions of the metabolic network of living cells. One way to ensure the continuous availability of reduced nicotinamide cofactors in a synthetic cell is to build a minimal enzymatic module that can oxidize an external electron donor and reduce NAD(P)+. In the diverse world of metabolism there is a plethora of potential electron donors and enzymes known from living organisms to provide reducing power to NAD(P)+ coenzymes. This perspective proposes guidelines to enable the reduction of nicotinamide cofactors enclosed in phospholipid vesicles, while avoiding high burdens of or cross-talk with other encapsulated metabolic modules. By determining key requirements, such as the feasibility of the reaction and transport of the electron donor into the cell-like compartment, we select a shortlist of potentially suitable electron donors. We review the most convenient proteins for the use of these reducing agents, highlighting their main biochemical and structural features. Noting that specificity toward either NAD(H) or NADP(H) imposes a limitation common to most of the analyzed enzymes, we discuss the need for specific enzymes─transhydrogenases─to overcome this potential bottleneck.

Paving the way for synthetic C1 - Metabolism in Pseudomonas putida through the reductive glycine pathway.

One-carbon (C1) compounds such as methanol, formate, and CO2 are alternative, sustainable microbial feedstocks for the biobased production of chemicals and fuels. In this study, we engineered the carbon metabolism of the industrially important bacterium Pseudomonas putida to modularly assimilate these three substrates through the reductive glycine pathway. First, we demonstrated the functionality of the C1-assimilation module by coupling the growth of auxotrophic strains to formate assimilation. Next, we extended the module in the auxotrophic strains from formate to methanol-dependent growth using both NAD and PQQ-dependent methanol dehydrogenases. Finally, we demonstrated, for the first time, engineered CO2-dependent formation of part of the biomass through CO2 reduction to formate by the native formate dehydrogenase, which required short-term evolution to rebalance the cellular NADH/NAD + ratio. This research paves the way to further engineer P. putida towards full growth on formate, methanol, and CO2 as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell factory.

Revealing determinants of translation efficiency via whole-gene codon randomization and machine learning.

It has been known for decades that codon usage contributes to translation efficiency and hence to protein production levels. However, its role in protein synthesis is still only partly understood. This lack of understanding hampers the design of synthetic genes for efficient protein production. In this study, we generated a synonymous codon-randomized library of the complete coding sequence of red fluorescent protein. Protein production levels and the full coding sequences were determined for 1459 gene variants in Escherichia coli. Using different machine learning approaches, these data were used to reveal correlations between codon usage and protein production. Interestingly, protein production levels can be relatively accurately predicted (Pearson correlation of 0.762) by a Random Forest model that only relies on the sequence information of the first eight codons. In this region, close to the translation initiation site, mRNA secondary structure rather than Codon Adaptation Index (CAI) is the key determinant of protein production. This study clearly demonstrates the key role of codons at the start of the coding sequence. Furthermore, these results imply that commonly used CAI-based codon optimization of the full coding sequence is not a very effective strategy. One should rather focus on optimizing protein production via reducing mRNA secondary structure formation with the first few codons.

Optimizing microbial networks through metabolic bypasses.

Metabolism has long been considered as a relatively stiff set of biochemical reactions. This somewhat outdated and dogmatic view has been challenged over the last years, as multiple studies exposed unprecedented plasticity of metabolism by exploring rational and evolutionary modifications within the metabolic network of cell factories. Of particular importance is the emergence of metabolic bypasses, which consist of enzymatic reaction(s) that support unnatural connections between metabolic nodes. Such novel topologies can be generated through the introduction of heterologous enzymes or by upregulating native enzymes (sometimes relying on promiscuous activities thereof). Altogether, the adoption of bypasses resulted in an expansion in the capacity of the host's metabolic network, which can be harnessed for bioproduction. In this review, we discuss modifications to the canonical architecture of central carbon metabolism derived from such bypasses towards six optimization purposes: stoichiometric gain, overcoming kinetic limitations, solving thermodynamic barriers, circumventing toxic intermediates, uncoupling product synthesis from biomass formation, and altering redox cofactor specificity. The metabolic costs associated with bypass-implementation are likewise discussed, including tailoring their design towards improving bioproduction.

Engineering the Reductive Glycine Pathway: A Promising Synthetic Metabolism Approach for C1-Assimilation.

In recent years the reductive glycine pathway (rGlyP) has emerged as a promising pathway for the assimilation of formate and other sustainable C1-feedstocks for future biotechnology. It was originally proposed as an attractive "synthetic pathway" to support formatotrophic growth due to its high ATP efficiency, linear structure, and limited overlap with native pathways in most microbial hosts. Here, we present the current state of research on this pathway including breakthroughs on its engineering. Different variants of the rGlyP are discussed, including its core module for formate to glycine conversion, as well as varying modules for substrate conversion to formate, and glycine assimilation routes. Very recently, the rGlyP has been successfully implemented for synthetic formatotrophic growth, as well as for growth on methanol, in some bacterial hosts. We discuss the engineering strategies employed in these studies, including growth-coupled selection of functional pathway modules. We also compare the rGlyP to other natural and synthetic C1-assimilation pathways. Finally, we provide an outlook on open challenges and opportunities for the rGlyP, including its engineering into more biotechnological hosts, as well as the still-to-be realized production of value-added chemicals via this pathway. We expect that further research on the rGlyP will support the efficient use of sustainable C1-substrates in bioproduction.

Towards a synthetic cell cycle.

Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.

Synthetic Biology Approaches To Enhance Microalgal Productivity.

The major bottleneck in commercializing biofuels and other commodities produced by microalgae is the high cost associated with phototrophic cultivation. Improving microalgal productivities could be a solution to this problem. Synthetic biology methods have recently been used to engineer the downstream production pathways in several microalgal strains. However, engineering upstream photosynthetic and carbon fixation metabolism to enhance growth, productivity, and yield has barely been explored in microalgae. We describe strategies to improve the generation of reducing power from light, as well as to improve the assimilation of CO2 by either the native Calvin cycle or synthetic alternatives. Overall, we are optimistic that recent technological advances will prompt long-awaited breakthroughs in microalgal research.

Reductive Glycine Pathway: A Versatile Route for One-Carbon Biotech.

Hong et al. heterologously expressed the metabolic core of the reductive glycine pathway (rGlyP) as a sink for the anaerobic conversion of glycerol. This recent study concludes several reports in 2020 on the ATP-efficient, one-carbon-assimilating rGlyP. Its engineering in diverse hosts could help the transformation toward renewable, one-carbon-based bioproduction.

The Ongoing Quest to Crack the Genetic Code for Protein Production.

Understanding the genetic design principles that determine protein production remains a major challenge. Although the key principles of gene expression were discovered 50 years ago, additional factors are still being uncovered. Both protein-coding and non-coding sequences harbor elements that collectively influence the efficiency of protein production by modulating transcription, mRNA decay, and translation. The influences of many contributing elements are intertwined, which complicates a full understanding of the individual factors. In natural genes, a functional balance between these factors has been obtained in the course of evolution, whereas for genetic-engineering projects, our incomplete understanding still limits optimal design of synthetic genes. However, notable advances have recently been made, supported by high-throughput analysis of synthetic gene libraries as well as by state-of-the-art biomolecular techniques. We discuss here how these advances further strengthen understanding of the gene expression process and how they can be harnessed to optimize protein production.

The reductive glycine pathway allows autotrophic growth of Desulfovibrio desulfuricans.

Six CO2 fixation pathways are known to operate in photoautotrophic and chemoautotrophic microorganisms. Here, we describe chemolithoautotrophic growth of the sulphate-reducing bacterium Desulfovibrio desulfuricans (strain G11) with hydrogen and sulphate as energy substrates. Genomic, transcriptomic, proteomic and metabolomic analyses reveal that D. desulfuricans assimilates CO2 via the reductive glycine pathway, a seventh CO2 fixation pathway. In this pathway, CO2 is first reduced to formate, which is reduced and condensed with a second CO2 to generate glycine. Glycine is further reduced in D. desulfuricans by glycine reductase to acetyl-P, and then to acetyl-CoA, which is condensed with another CO2 to form pyruvate. Ammonia is involved in the operation of the pathway, which is reflected in the dependence of the autotrophic growth rate on the ammonia concentration. Our study demonstrates microbial autotrophic growth fully supported by this highly ATP-efficient CO2 fixation pathway.

Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle.

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term "phosphoglycolate salvage." Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2 We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2 We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.

Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator.

Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant hosts that can grow on formate, but it uses the Calvin cycle, the high ATP cost of which limits biomass and product yields. Here, we redesign C. necator metabolism for formate assimilation via the synthetic, highly ATP-efficient reductive glycine pathway. First, we demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By integrating glycine biosynthesis and assimilation we are able to replace C. necator's Calvin cycle with the synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism and use short-term evolution to optimize pathway activity. The final growth yield we achieve (2.6 gCDW/mole-formate) nearly matches that of the WT strain using the Calvin Cycle (2.9 gCDW/mole-formate). We expect that further rational and evolutionary optimization will result in a superior formatotrophic C. necator strain, paving the way towards realizing the formate bio-economy.

Renewable methanol and formate as microbial feedstocks.

Methanol and formate are attractive microbial feedstocks as they can be sustainably produced from CO2 and renewable energy, are completely miscible, and are easy to store and transport. Here, we provide a biochemical perspective on microbial growth and bioproduction using these compounds. We show that anaerobic growth of acetogens on methanol and formate is more efficient than on H2/CO2 or CO. We analyze the aerobic C1 assimilation pathways and suggest that new-to-nature routes could outperform their natural counterparts. We further discuss practical bioprocessing aspects related to growth on methanol and formate, including feedstock toxicity. While challenges in realizing sustainable production from methanol and formate still exist, the utilization of these feedstocks paves the way towards a truly circular carbon economy.

Bicistronic Design-Based Continuous and High-Level Membrane Protein Production in Escherichia coli.

Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.

A critical comparison of cellular and cell-free bioproduction systems.

Conversion of biological feedstocks into value-added chemicals is mostly performed via microbial fermentation. An emerging alternative approach is the use of cell-free systems, consisting of purified enzymes and cofactors. Unfortunately, the in vivo and in vitro research communities rarely interact, which leads to oversimplifications and exaggerations that do not permit fair comparison of the two strategies and impede synergistic interactions. Here, we provide a comprehensive account for the advantages and drawbacks associated with each strategy, and further discuss recent research efforts that aim to breach the limits of cellular and cell-free production. We also explore emerging hybrid solutions that integrate the benefits of both worlds and could expand the boundaries of biosynthesis.