Automated in vivo enzyme engineering accelerates biocatalyst optimization.

Achieving cost-competitive bio-based processes requires development of stable and selective biocatalysts. Their realization through in vitro enzyme characterization and engineering is mostly low throughput and labor-intensive. Therefore, strategies for increasing throughput while diminishing manual labor are gaining momentum, such as in vivo screening and evolution campaigns. Computational tools like machine learning further support enzyme engineering efforts by widening the explorable design space. Here, we propose an integrated solution to enzyme engineering challenges whereby ML-guided, automated workflows (including library generation, implementation of hypermutation systems, adapted laboratory evolution, and in vivo growth-coupled selection) could be realized to accelerate pipelines towards superior biocatalysts.

Synergistic investigation of natural and synthetic C1-trophic microorganisms to foster a circular carbon economy.

A true circular carbon economy must upgrade waste greenhouse gases. C1-based biomanufacturing is an attractive solution, in which one carbon (C1) molecules (e.g. CO2, formate, methanol, etc.) are converted by microbial cell factories into value-added goods (i.e. food, feed, and chemicals). To render C1-based biomanufacturing cost-competitive, we must adapt microbial metabolism to perform chemical conversions at high rates and yields. To this end, the biotechnology community has undertaken two (seemingly opposing) paths: optimizing natural C1-trophic microorganisms versus engineering synthetic C1-assimilation de novo in model microorganisms. Here, we pose how these approaches can instead create synergies for strengthening the competitiveness of C1-based biomanufacturing as a whole.

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.

Emergent CRISPR-Cas-based technologies for engineering non-model bacteria.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) technologies brought a transformative change in the way bacterial genomes are edited, and a plethora of studies contributed to developing multiple tools based on these approaches. Prokaryotic biotechnology benefited from the implementation of such genome engineering strategies, with an increasing number of non-model bacterial species becoming genetically tractable. In this review, we summarize the recent trends in engineering non-model microbes using CRISPR-Cas technologies, discussing their potential in supporting cell factory design towards biotechnological applications. These efforts include, among other examples, genome modifications as well as tunable transcriptional regulation (both positive and negative). Moreover, we examine how CRISPR-Cas toolkits for engineering non-model organisms enabled the exploitation of emergent biotechnological processes (e.g. native and synthetic assimilation of one-carbon substrates). Finally, we discuss our slant on the future of bacterial genome engineering for domesticating non-model organisms in light of the most recent advances in the ever-expanding CRISPR-Cas field.

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.

The transition of Rhodobacter sphaeroides into a microbial cell factory.

Microbial cell factories are the workhorses of industrial biotechnology and improving their performances can significantly optimize industrial bioprocesses. Microbial strain engineering is often employed for increasing the competitiveness of bio-based product synthesis over more classical petroleum-based synthesis. Recently, efforts for strain optimization have been standardized within the iterative concept of "design-build-test-learn" (DBTL). This approach has been successfully employed for the improvement of traditional cell factories like Escherichia coli and Saccharomyces cerevisiae. Within the past decade, several new-to-industry microorganisms have been investigated as novel cell factories, including the versatile α-proteobacterium Rhodobacter sphaeroides. Despite its history as a laboratory strain for fundamental studies, there is a growing interest in this bacterium for its ability to synthesize relevant compounds for the bioeconomy, such as isoprenoids, poly-ß-hydroxybutyrate, and hydrogen. In this study, we reflect on the reasons for establishing R. sphaeroides as a cell factory from the perspective of the DBTL concept. Moreover, we discuss current and future opportunities for extending the use of this microorganism for the bio-based economy. We believe that applying the DBTL pipeline for R. sphaeroides will further strengthen its relevance as a microbial cell factory. Moreover, the proposed use of strain engineering via the DBTL approach may be extended to other microorganisms that have not been critically investigated yet for industrial applications.

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides.

BACKGROUND: Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-ß-hydroxybutyrate (PHB) biosynthesis. RESULTS: This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed 13C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways. CONCLUSIONS: Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.

CRISPR with a Happy Ending: Non-Templated DNA Repair for Prokaryotic Genome Engineering.

The exploration of microbial metabolism is expected to support the development of a sustainable economy and tackle several problems related to the burdens of human consumption. Microorganisms have the potential to catalyze processes that are currently unavailable, unsustainable and/or inefficient. Their metabolism can be optimized and further expanded using tools like the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) systems. These tools have revolutionized the field of biotechnology, as they greatly streamline the genetic engineering of organisms from all domains of life. CRISPR-Cas and other nucleases mediate double-strand DNA breaks, which must be repaired to prevent cell death. In prokaryotes, these breaks can be repaired through either homologous recombination, when a DNA repair template is available, or through template-independent end joining, of which two major pathways are known. These end joining pathways depend on different sets of proteins and mediate DNA repair with different outcomes. Understanding these DNA repair pathways can be advantageous to steer the results of genome engineering experiments. In this review, we discuss different strategies for the genetic engineering of prokaryotes through either non-homologous end joining (NHEJ) or alternative end joining (AEJ), both of which are independent of exogenous DNA repair templates.

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides.

Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4-13 C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.

Metabolic flux ratio analysis by parallel 13C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides.

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from 13C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-13C]- and [4-13C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer 13C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.

Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering.

BACKGROUND: Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-ß-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. RESULTS: Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected. CONCLUSIONS: In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

Characterization of heterotrophic growth and sesquiterpene production by Rhodobacter sphaeroides on a defined medium.

Rhodobacter sphaeroides is a metabolically versatile bacterium capable of producing terpenes natively. Surprisingly, terpene biosynthesis in this species has always been investigated in complex media, with unknown compounds possibly acting as carbon and nitrogen sources. Here, a defined medium was adapted for R. sphaeroides dark heterotrophic growth, and was used to investigate the conversion of different organic substrates into the reporter terpene amorphadiene. The amorphadiene synthase was cloned in R. sphaeroides, allowing its biosynthesis via the native 2-methyl-D-erythritol-4-phosphate (MEP) pathway and, additionally, via a heterologous mevalonate one. The latter condition increased titers up to eightfold. Consequently, better yields and productivities to previously reported complex media cultivations were achieved. Productivity was further investigated under different cultivation conditions, including nitrogen and oxygen availability. This novel cultivation setup provided useful insight into the understanding of terpene biosynthesis in R. sphaeroides, allowing to better comprehend its dynamics and regulation during chemoheterotrophic cultivation.

Heterologous expression of Mus musculus immunoresponsive gene 1 (irg1) in Escherichia coli results in itaconate production.

Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalyzed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1), was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in Escherichia coli with production titres up to 560 mg/L.