Metabolic engineering of Pichia pastoris for malic acid production from methanol

Abstract

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to byproduct generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.

Metabolic Engineering of the Methylotrophic Yeast Pichia Pastoris (Komagataella phaffii) for the Production of Β-alanine From Methanol

“…The coding sequences were individually placed under the control of the strong AOX1 promoters and integrated into the genome of P. pastoris. In order to increase the e ciency of homologous recombination (HR), the KU70 mutant strain (Δku70) which has impaired nonhomologous end joining (NHEJ) was used as a parent strain (Guo et al 2021;Weninger et al 2018). The individual overexpression of ADCs from B. paralicheniformis, B. subtilis and C. glutamicum enabled the accumulation of β-alanine after 6 d of fermentation when feeding the strains with methanol (Fig.…”

“…Reports on metabolite production in fermentations of engineered P. pastoris mainly remain at the proof-of-concept stage, with titers usually lower than 1 g/L, especially when methanol is used as substrate (Gao et al 2021). Several recent studies reported gram per liter metabolites production in shake asks, with examples including isobutanol (Siripong et al 2020;Siripong et al 2018), malic acid (Guo et al 2021), and D-lactic acid (Yamada et al 2019). Unfortunately, the chemicals production potential of these recombinant P. pastoris strains was not fully evaluated by cultivation on the fermenter scale.…”

“…The KU70 gene of the wild-type P. pastoris strain GS115 was deleted using the Cre-LoxP method to improve the homologous recombination e ciency (Guo et al 2021;Weninger et al 2018). The expression vectors pMPICZHis-bliPanD, pMPICZHis-bsPanD, pMPICZHis-sgePanD, pMPICZHis-cguPanD, pMPICZHis-TcCSADC, pMPICZHis-ADC-Oan, pMPICZHis-ADC-Spe, pMPICZHis-2ADC, and pMPICZHis-2ADC-Spe were linearized with BspEI and used to transform the Δku70 strain by electroporation.…”

“…Methanol is considered one of the most promising feedstocks due to its unique advantages such as providing highly reduced carbon, not competing with food sources, and potentially sustainable production in the future (Zhu et al 2020). Consequently, there is an emerging trend of utilizing methanol as an alternative feedstock for chemical production using natural or synthetic methylotrophs (Guo et al 2021;Jin et al 2021;Tuyishime et al 2018;Whitaker et al 2017;Zhu et al 2016).…”

Biocatalytic C-C Bond Formation for One Carbon Resource Utilization

“…Currently, DAS is not exploited as a biocatalyst. However, Pichia pastoris , a kind of methylotrophic yeast, have been engineered to produce chemicals from methanol using XuMP [ 81 ]. Similar to DAS, transketolase (TK, EC 2.2.1.1) can catalyze the reaction of D-fructose-6-phosphate or L-sorbose with formaldehyde to generate dihydroxyacetone [ 82 ].…”