The increase of CO
2
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 CO
2
as a feedstock gains an extensive importance to establish CO
2
-neutral or CO
2
-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 CO
2
as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis.
13
C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO
2
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 CO
2
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 CO
2
, to support sustainability of industrial bioprocesses.
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.
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