A synthetic methylotrophic Escherichia coli as a chassis for bioproduction from methanol.

Methanol synthesized from captured greenhouse gases is an emerging renewable feedstock with great potential for bioproduction. Recent research has raised the prospect of methanol bioconversion to value-added products using synthetic methylotrophic Escherichia coli, as its metabolism can be rewired to enable growth solely on the reduced one-carbon compound. Here we describe the generation of an E. coli strain that grows on methanol at a doubling time of 4.3 h-comparable to many natural methylotrophs. To establish bioproduction from methanol using this synthetic chassis, we demonstrate biosynthesis from four metabolic nodes from which numerous bioproducts can be derived: lactic acid from pyruvate, polyhydroxybutyrate from acetyl coenzyme A, itaconic acid from the tricarboxylic acid cycle and p-aminobenzoic acid from the chorismate pathway. In a step towards carbon-negative chemicals and valorizing greenhouse gases, our work brings synthetic methylotrophy in E. coli within reach of industrial applications.

Conversion of CO2 into organic acids by engineered autotrophic yeast.

The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L-1, while balancing the expression of key genes and modifying screening conditions led to 2 g L-1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses.

Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle.

Methanol is a liquid with high energy storage capacity that holds promise as an alternative substrate to replace sugars in the biotechnology industry. It can be produced from CO2 or methane and its use does not compete with food and animal feed production. However, there are currently only limited biotechnological options for the valorization of methanol, which hinders its widespread adoption. Here, we report the conversion of the industrial platform organism Escherichia coli into a synthetic methylotroph that assimilates methanol via the energy efficient ribulose monophosphate cycle. Methylotrophy is achieved after evolution of a methanol-dependent E. coli strain over 250 generations in continuous chemostat culture. We demonstrate growth on methanol and biomass formation exclusively from the one-carbon source by 13C isotopic tracer analysis. In line with computational modeling, the methylotrophic E. coli strain optimizes methanol oxidation by upregulation of an improved methanol dehydrogenase, increasing ribulose monophosphate cycle activity, channeling carbon flux through the Entner-Doudoroff pathway and downregulating tricarboxylic acid cycle enzymes. En route towards sustainable bioproduction processes, our work lays the foundation for the efficient utilization of methanol as the dominant carbon and energy resource.

Adaptive laboratory evolution and reverse engineering enhances autotrophic growth in Pichia pastoris.

Synthetic biology offers several routes for CO2 conversion into biomass or bio-chemicals, helping to avoid unsustainable use of organic feedstocks, which negatively contribute to climate change. The use of well-known industrial organisms, such as the methylotrophic yeast Pichia pastoris (Komagataella phaffii), for the establishment of novel C1-based bioproduction platforms could wean biotechnology from feedstocks with alternative use in food production. Recently, the central carbon metabolism of P. pastoris was re-wired following a rational engineering approach, allowing the resulting strains to grow autotrophically with a µmax of 0.008 h-1, which was further improved to 0.018 h-1 by adaptive laboratory evolution. Using reverse genetic engineering of single-nucleotide (SNPs) polymorphisms occurring in the genes encoding for phosphoribulokinase and nicotinic acid mononucleotide adenylyltransferase after evolution, we verified their influence on the improved autotrophic phenotypes. The reverse engineered SNPs lead to lower enzyme activities in putative branching point reactions and in reactions involved in energy balancing. Beyond this, we show how further evolution facilitates peroxisomal import and increases growth under autotrophic conditions. The engineered P. pastoris strains are a basis for the development of a platform technology, which uses CO2 for production of value-added products, such as cellular biomass, technical enzymes and chemicals and which further avoids consumption of organic feedstocks with alternative use in food production. Further, the identification and verification of three pivotal steps may facilitate the integration of heterologous CBB cycles or similar pathways into heterotrophic organisms.

The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2.

The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO2. By addition of eight heterologous genes and deletion of three native genes, we engineer the peroxisomal methanol-assimilation pathway of P. pastoris into a CO2-fixation pathway resembling the Calvin-Benson-Bassham cycle, the predominant natural CO2-fixation pathway. The resulting strain can grow continuously with CO2 as a sole carbon source at a µmax of 0.008 h-1. The specific growth rate was further improved to 0.018 h-1 by adaptive laboratory evolution. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO2, and by avoiding consumption of an organic feedstock with alternative uses in food production.

CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris.

State-of-the-art strain engineering techniques for the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) include overexpression of endogenous and heterologous genes and deletion of host genes. For efficient gene deletion, methods such as the split-marker technique have been established. However, synthetic biology trends move toward building up large and complex reaction networks, which often require endogenous gene knockouts and simultaneous overexpression of individual genes or whole pathways. Realization of such engineering tasks by conventional approaches employing subsequent steps of transformations and marker recycling is very time- and labor-consuming. Other applications require tagging of certain genes/proteins or promoter exchange approaches, which are hard to design and construct with conventional methods. Therefore, efficient systems are required that allow precise manipulations of the P. pastoris genome, including simultaneous overexpression of multiple genes. To meet this challenge, we have developed a CRISPR/Cas9-based kit for gene insertions, deletions, and replacements, which paves the way for precise genomic modifications in P. pastoris. In this chapter, the versatile method for performing these modifications without the integration of a selection marker is described. A ready-to-use plasmid kit for performing CRISPR/Cas9-mediated genome editing in P. pastoris based on the GoldenPiCS modular cloning vectors is available at Addgene as CRISPi kit (#1000000136).

GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris.

BACKGROUND: State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in P. pastoris. However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for P. pastoris. METHODS: With the GoldenPiCS system, we provide a flexible modular system for advanced strain engineering in P. pastoris based on Golden Gate cloning. For this purpose, we established a wide variety of standardized genetic parts (20 promoters of different strength, 10 transcription terminators, 4 genome integration loci, 4 resistance marker cassettes). RESULTS: All genetic parts were characterized based on their expression strength measured by eGFP as reporter in up to four production-relevant conditions. The promoters, which are either constitutive or regulatable, cover a broad range of expression strengths in their active conditions (2-192% of the glyceraldehyde-3-phosphate dehydrogenase promoter P GAP ), while all transcription terminators and genome integration loci led to equally high expression strength. These modular genetic parts can be readily combined in versatile order, as exemplified for the simultaneous expression of Cas9 and one or more guide-RNA expression units. Importantly, for constructing multigene constructs (vectors with more than two expression units) it is not only essential to balance the expression of the individual genes, but also to avoid repetitive homologous sequences which were otherwise shown to trigger "loop-out" of vector DNA from the P. pastoris genome. CONCLUSIONS: GoldenPiCS, a modular Golden Gate-derived P. pastoris cloning system, is very flexible and efficient and can be used for strain engineering of P. pastoris to accomplish pathway expression, protein production or other applications where the integration of various DNA products is required. It allows for the assembly of up to eight expression units on one plasmid with the ability to use different characterized promoters and terminators for each expression unit. GoldenPiCS vectors are available at Addgene.