Low carbon and excessive production of cordycepin by Pichia pastoris cell factory

Abstract

Cordycepin is a nucleoside analogue that is widely used in medicine and health products.

The production of cordycepin from Cordyceps militaris faces the challenges of low productivity and high greenhouse gas emission rate.

In this study, the cordycepin high-producing strain Pp29 was constructed by optimizing the cordycepin biosynthesis pathway through promoter combination, Kozak sequence and enzyme fusion, enhancing the methanol assimilation capacity in peroxisome, regulating the synthesis of NADPH and ATP, and enhancing the supply of adenosine and 3'-AMP. It can produce 1551.44 mg/L cordycepin by shaking bottle fermentation.

Pp29 achieved the highest yield to date (8.11 g/L) by fed-batch fermentation in a 10 L fermenter, with CO2 equivalent emissions 1.9-17.3 times lower than Cordyceps pupa and other yeast systems.

This study provided the basis for the synthesis of cordycepin and other nucleoside analogues from methanol in Pichia pastoris chassis cells.

Main content

1. Construction and optimization of cordycepin biosynthesis pathway of Pichia pastoris

After codon optimization, Ppcns1, Ppcns2 and Ppcns3/NK genes of Cordyceps militaris CP1 were integrated into Pichia Pastoris genome to construct the engineered strain of cordyceps.

Pichia Pastoris GS115 showed strong tolerance to cordycepin and was suitable for production of cordycepin.

Kozak sequence plays a key role in regulating transcription levels of downstream genes.

Among the eight Kozak sequences, the highest relative fluorescence intensity was detected after EGFP fusion with K7 sequence (CGGACC).

The K7 sequence was selected to be upstream of the start codon of all target genes.

The authors also optimized the promoter of the target gene, the selected linker for fusion expression of key enzymes and the copy number of the gene, and selected the strain Pp07 with the best cordycepin production for subsequent optimization.

Figure 1. Construction of cordycepin biosynthetic pathway of Pichia pastoris.

(A) Schematic diagram of cordycepin synthesis in the cytoplasm of engineered Pichia Pastoris.
(B) Inhibitory effect of cordycepin on the growth of Pichia GS115.
(C) The expression intensity of downstream genes was enhanced by optimizing Kozak sequence.
(D) Relative fluorescence intensity of EGFP controlled by different Kozak sequences.
(E) Construction and optimization of cordycepin biosynthesis pathway.
(F) Chromatogram and mass spectrometry of cordycepin and adenosine in standards and fermentation fluids.

2. Enhance methanol assimilation to increase carbon metabolism supply

Methanol is not only the inducer but also the only carbon source during the accumulation of cordycepin. The assimilation of methanol has an important effect on the growth of Pichia Pastoris and the overproduction of cordycepin.

Genes related to methanol assimilation efficiency were overexpressed. CAT and AOX1 were integrated into Pp07 genome in the form of N-CAT-G4S- Aox1-per2-C and localized to peroxisome. The cordycepin production of strain Pp15 was significantly increased, reaching 1209.7 mg/L. Compared with strain Pp07, it increased by 12.7%.

When the methanol concentration increased from 0.5% to 1%, the cordycepin production of strain Pp07 increased by 9.6%, and the cordycepin production of strain Pp15 increased by 10.5%.

When methanol concentration increased from 1% to 1.5%, cordycepin production of strain Pp07 decreased significantly by 14.0%, while strain Pp15 did not change significantly.

These results showed that strain Pp15 exhibited higher methanol tolerance than strain Pp07, 1% methanol was sufficient to support the necessary carbon metabolic flux, and the bottleneck of cordycepin synthesis was located downstream of the methanol assimilation pathway.

Figure 2. Peroxisome and enzyme fusion engineering enhance methanol assimilation and tolerance.

(A) Schematic diagram of peroxisome methanol assimilation promoted by enzyme fusion engineering and cordycepin yield change.
(B) 3D model prediction of fusion protein N-CAT-G4S-AOX1-per2-C.
(C) Cordycepin yield of strain Pp07 at 0.5-3% methanol concentration.
(D) Cordycepin yield of strain Pp15 at 0.5-3% methanol concentration.

3. Enhancing intracellular NADPH and ATP supply

Overexpression of PpGapN and PGK in strain Pp15 enhanced intracellular NADPH and ATP supply, while overexpression of PurF enhanced the supply of precursors, thus increasing the production of cordycepin in strain Pp25 to 1395.6 mg/L.

The intracellular NADPH and ATP levels of strains Pp15 and Pp25 were monitored every 24 h, and the NADPH and ATP contents of strains Pp25 were increased by 14.3-219.5% and 30.2-65.6%, respectively, compared with Pp15.

4. Increase the supply of direct precursors for cordycepin biosynthesis

Strain Pp29 was obtained through overexpression of ADSS and PpCpdB in strain Pp25, and the output of cordycepin increased to 1551.4 mg/L.

Monitoring the adenosine content in strains Pp25 and Pp29 every 24 h showed that adenosine content in Pp29 increased by 8.5-795.9% compared with Pp25, indicating that adequate precursor supply can promote cordycepin synthesis.

5. High density fermentation of cordycepin yield

The strain Pp29 was used for fed-batch fermentation in a 10 L fermenter.

When fermentation was terminated after 144 h, the yield of cordycepin reached 8108.3 mg/L, the productivity was 1351.4 mg/L/d, and the conversion rate of methanol to cordycepin was 25.7 mg/g.

Sum up

In this study, the cordycepin production was increased by 31.6%, 12.7%, 15.8% and 11.1%, respectively, by optimizing the cordycepin biosynthesis pathway, improving the methanol assimilation capacity, regulating ATP and NADPH synthesis and enhancing the supply of precursor substances.

The cordycepin high-yielding strain Pp29 achieved the highest yield to date of 8108.3 mg/L by fed-batch fermentation in a 10 L fermenter.