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.

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.

Single carbon metabolism - A new paradigm for microbial bioprocesses?

Increasing atmospheric carbon dioxide levels, a reduction of arable land area and the dependence of first and second generation biotechnology feedstocks on agricultural products, call for alternative, sustainable feedstock sources for industrial applications. The direct use of CO2 or conversion of CO2 into other single carbon (C1) sources have great potential as they might help to reduce carbon emissions and do not compete with agricultural land use. Here we discuss the microbial use of C1 carbon sources, their potential applications in biotechnology, and challenges towards sustainable C1-based industrial biotechnology processes. We focus on methanol, formic acid, methane, syngas, and CO2 as feedstocks for bioprocesses, their assimilation pathways, current and emerging applications, and limitations of their application. This mini-review is intended as a first introduction for researchers who are new to the field of C1 biotechnology.

A native phosphoglycolate salvage pathway of the synthetic autotrophic yeast Komagataella phaffii.

Synthetic autotrophs can serve as chassis strains for bioproduction from CO2 as a feedstock to take measures against the climate crisis. Integration of the Calvin-Benson-Bassham (CBB) cycle into the methylotrophic yeast Komagataella phaffii (Pichia pastoris) enabled it to use CO2 as the sole carbon source. The key enzyme in this cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzing the carboxylation step. However, this enzyme is error prone to perform an oxygenation reaction leading to the production of toxic 2-phosphoglycolate. Native autotrophs have evolved different recycling pathways for 2-phosphoglycolate. However, for synthetic autotrophs, no information is available for the existence of such pathways. Deletion of CYB2 in the autotrophic K. phaffii strain led to the accumulation of glycolate, an intermediate in phosphoglycolate salvage pathways, suggesting that such a pathway is enabled by native K. phaffii enzymes. 13C tracer analysis with labeled glycolate indicated that the yeast pathway recycling phosphoglycolate is similar to the plant salvage pathway. This orthogonal yeast pathway may serve as a sensor for RuBisCO oxygenation, and as an engineering target to boost autotrophic growth rates in K. phaffii.

The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO2 in the yeast Komagataella phaffii.

The current climatic change is predominantly driven by excessive anthropogenic CO2 emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO2 co-assimilation or the use of CO2-derived methanol or formate as carbon sources are considered pathbreaking contributions to solving this global problem. The number of industrially-relevant microorganisms that can use these two carbon sources is limited, and even fewer can concurrently co-assimilate CO2. Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO2 in the industrially-relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using 13C-tracer-based metabolomic techniques and metabolic engineering approaches, we discover and confirm a growth supporting pathway based on native enzymes that can perform all three assimilations: namely, the oxygen-tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO2 utilisation to produce proteins, biomass, or chemicals in yeast.

Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA Scaffolds.

For metabolic engineering approaches, fast and reliable tools are required to precisely manipulate the expression of target genes. dCas9 can be fused via RNA scaffolds to trans-activator domains and thus regulate the gene expression when targeted to the promoter region of a gene. In this work we show that this strategy can be successfully implemented for the methylotrophic yeast Pichia pastoris. It is shown that the thiamine repressible promoter of THI11 can be activated under repression conditions using a scgRNA/dCas9 construct. Furthermore, the RIB1 gene required for riboflavin production was activated, leading to increased riboflavin production exceeding the riboflavin titers of a conventional RIB1 overexpression with a pGAP promoter.