Summary
Methanol biotransformation can expand biorefinery substrate spectrum other than biomass by using methylotrophic microbes.
Ogataea
(
Hansenula
)
polymorpha
, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but the low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR-Cas9 system in
O. polymorpha
. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ) increased HR rates from 20%–30% to 60%–70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated system has been applied for homologous integration of large fragments and
in vivo
assembly of multiple fragments, which enables the production of fatty alcohols in
O. polymorpha
. These findings will simplify genetic engineering in non-conventional yeasts and facilitate the adoption of
O. polymorpha
as an attractive cell factory for industrial application.
Methylotrophic yeast
Ogataea polymorpha
is capable to utilize multiple carbon feedstocks especially methanol as sole carbon source and energy, making it an ideal host for bio-manufacturing. However, the lack of gene integration sites limits its systems metabolic engineering, in particular construction of genome-integrated pathway. We here screened the genomic neutral sites for gene integration without affecting cellular fitness, by genomic integration of an enhanced green fluorescent protein (
eGFP
) gene via CRISPR-Cas9 technique. After profiling the growth and fluorescent intensity in various media, 17 genome positions were finally identified as potential neutral sites. Finally, integration of fatty alcohol synthetic pathway genes into neutral sites NS2 and NS3, enabled the production of 4.5 mg/L fatty alcohols, indicating that these neutral sites can be used for streamline metabolic engineering in
O. polymorpha
. We can anticipate that the neutral sites screening method described here can be easily adopted to other eukaryotes.
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