Microbial production of α-farnesene from renewable raw materials is a feasible alternative to traditional petroleum craft. Recently, the research on improving α-farnesene production in Pichia pastoris mainly focused on cytoplasmic engineering, while comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we first sought to confirm that the isopentenol utilization pathway (IUP) could act as a two-step shortcut for IPP synthesis in P. pastoris peroxisomes. In addition, we proposed dual regulation of cytoplasm and peroxisomes to boost α-farnesene synthesis in P. pastoris X33, thus the resultant strain produced 2.18 ± 0.04 g/L, which was 1.3 times and 2.1 times than that of the strain only with peroxisomal or cytoplasmic engineering, respectively. The α-farnesene production achieved 2.56 ± 0.04 g/L in shake flasks after carbon source cofeeding, which was the highest reported production in worldwide literatures to the best of my knowledge. Therefore, we propose these strategies as efficient approaches to enhancing α-farnesene production in P. pastoris, which might bring new ideas for the biosynthesis of high-value compounds.

α-Farnesene, an acyclic volatile sesquiterpene, plays important roles in aircraft fuel, food flavoring, agriculture, pharmaceutical and chemical industries. Here, by re-creating the NADPH and ATP biosynthetic pathways in Pichia pastoris, we increased the production of α-farnesene. First, the native oxiPPP was recreated by overexpressing its essential enzymes or by inactivating glucose-6-phosphate isomerase (PGI). This revealed that the combined over-expression of ZWF1 and SOL3 increases α-farnesene production by improving NADPH supply, whereas inactivating PGI did not do so because it caused a reduction in cell growth. The next step was to introduce heterologous cPOS5 at various expression levels into P. pastoris. It was discovered that a low intensity expression of cPOS5 aided in the production of α-farnesene. Finally, ATP was increased by the overexpression of APRT and inactivation of GPD1. The resultant strain P. pastoris X33-38 produced 3.09 ± 0.37 g/L of α-farnesene in shake flask fermentation, which was 41.7% higher than that of the parent strain. These findings open a new avenue for the development of an industrial-strength α-farnesene producer by rationally modifying the NADPH and ATP regeneration pathways in P. pastoris.

The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMI Cg ::I Sm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDH Cg ) by NADP-GapDH Ca from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ signi cantly between these strains (14.1 ± 1.8 g/L v.s. 16.2 ± 1.1 g/L). Enhancing the carbon ux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icd Cg were introduced into C. glutamicum with NADP-GapDH Ca and pEC-ABNCE increased L-leucine yield (32.8 ± 1.7 g/L) and improved cell growth (OD 562 = 41.3 ± 4.2) because the resultant strain C. glutamicum XL-1 ΔMI Cg ::I Sm G Cg ::G Ca P zwf -D1 P icd -D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the rst report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.

α-Farnesene, an acyclic volatile sesquiterpene, plays important roles in aircraft fuel, food flavoring, agriculture, pharmaceutical and chemical industries. Here, we enhanced α-farnesene production through reconstructing the biosynthetic pathways of NADPH and ATP in Pichia pastoris. First, the native oxiPPP was reconstructed by over-expressing the key enzymes in oxiPPP or/and inactivating glucose-6-phosphate isomerase (PGI), indicating that combined over-expression of ZWF1 and SOL3 improves NADPH supply and thus increasing α-farnesene production while inactivation of PGI was not because of the decreased cell growth. Next, different expression level of heterologous cPOS5 were introduced into P. pastoris, and found that low intensity expression of cPOS5 facilitated α-farnesene biosynthesis. Finally, ATP was increased by overexpression of APRT and inactivation of GPD1. The resultant strain P. pastoris X33-38 produced 3.09±0.37 g/L of α-farnesene in shake-flask fermentation, which was 41.7% higher than that of the parent strain. These results provide a new perspective to construct industrial-strength α-farnesene producer by rational modification of NADPH and ATP regeneration pathway in P. pastoris.

The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMICg::ISm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDHCg) by NADP-GapDHCa from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ significantly between these strains (14.1±1.8 g/L v.s. 16.2±1.1 g/L). Enhancing the carbon flux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icdCg were introduced into C. glutamicum with NADP-GapDHCa and pEC-ABNCE increased L-leucine yield (32.8±1.7 g/L) and improved cell growth (OD562=41.3±4.2) because the resultant strain C. glutamicum XL-1 ΔMICg::ISm GCg::GCa Pzwf-D1 Picd-D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the first report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.