Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH.

Currently, the biological production of L-malic acid (L-MA) is mainly based on the fermentation of filamentous fungi at near-neutral pH, but this process requires large amounts of neutralizing agents, resulting in the generation of waste salts when free acid is obtained in the downstream process, and the environmental hazards associated with the waste salts limit the practical application of this process. To produce L-MA in a more environmentally friendly way, we metabolically engineered the acid-tolerant yeast Pichia kudriavzevii and achieved efficient production of L-MA through low pH fermentation. First, an initial L-MA-producing strain that relies on the reductive tricarboxylic acid (rTCA) pathway was constructed. Subsequently, the L-MA titer and yield were further increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. In addition, we found that the insufficient supply of NADH for cytoplasmic malate dehydrogenase (MDH) hindered the L-MA production at low pH, which was resolved by overexpressing the soluble pyridine nucleotide transhydrogenase SthA from E. coli. Transcriptomic and metabolomic data showed that overexpression of EcSthA contributed to the activation of the pentose phosphate pathway and provided additional reducing power for MDH by converting NADPH to NADH. Furthermore, overexpression of EcSthA was found to help reduce the accumulation of the by-product pyruvate but had no effect on the accumulation of succinate. In microaerobic batch fermentation in a 5-L fermenter, the best strain, MA009-10-URA3 produced 199.4 g/L L-MA with a yield of 0.94 g/g glucose (1.27 mol/mol), with a productivity of 1.86 g/L/h. The final pH of the fermentation broth was approximately 3.10, meaning that the amount of neutralizer used was reduced by more than 50% compared to the common fermentation processes using filamentous fungi. To our knowledge, this is the first report of the efficient bioproduction of L-MA at low pH and represents the highest yield of L-MA in yeasts reported to date.

Identification of Absidia orchidis steroid 11ß-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone.

Hydrocortisone is an effective anti-inflammatory drug and also an important intermediate for synthesis of other steroid drugs. The filamentous fungus Absidia orchidis is renowned for biotransformation of acetylated cortexolone through 11ß-hydroxylation to produce hydrocortisone. However, due to the presence of 11α-hydroxylase in A. orchidis, the 11α-OH by-product epi-hydrocortisone is always produced in a 1:1 M ratio with hydrocortisone. In order to decrease epi-hydrocortisone production, Saccharomyces cerevisiae was engineered in this work as an alternative way to produce hydrocortisone through biotransformation. Through transcriptomic analysis coupled with genetic verification in S. cerevisiae, the A. orchidis steroid 11ß-hydroxylation system was characterized, including a cytochrome P450 enzyme CYP5311B2 and its associated redox partners cytochrome P450 reductase and cytochrome b5. CYP5311B2 produces a mix of stereoisomers containing 11ß- and 11α-hydroxylation derivatives in a 4:1 M ratio. This fungal steroid 11ß-hydroxylation system was reconstituted in S. cerevisiae for hydrocortisone production, resulting in a productivity of 22 mg/L·d. Protein engineering of CYP5311B2 generated a R126D/Y398F variant, which had 3 times higher hydrocortisone productivity compared to the wild type. Elimination of C20-hydroxylation by-products and optimization of the expression of A. orchidis 11ß-hydroxylation system genes further increased hydrocortisone productivity by 238% to 223 mg/L·d. In addition, a novel steroid transporter ClCDR4 gene was identified from Cochliobolus lunatus, overexpression of which further increased hydrocortisone productivity to 268 mg/L·d in S. cerevisiae. Through increasing cell mass, 1060 mg/L hydrocortisone was obtained in 48 h and the highest productivity reached 667 mg/L·d. This is the highest hydrocortisone titer reported for yeast biotransformation system so far.

Production of 14α-hydroxysteroids by a recombinant Saccharomyces cerevisiae biocatalyst expressing of a fungal steroid 14α-hydroxylation system.

The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11ß-/14α-hydroxylase P-450lun from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11ß-hydroxylation activity of the P-450lun towards cortexolone (RSS) substrate; however, the potential steroid 14α-hydroxylation activity of this enzyme, especially for androstenedione (AD) substrate, has not yet conducted in-depth testing. In this work, we further tested the steroid 14α-hydroxylation activity of the P-450lun towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450lun functions as the specific 14α-hydroxylase towards the AD substrate (regiospecificity > 99%); however, it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition, through transcriptome analysis combined with gene functional characterizations, we also identified and cloned the gene for the P-450lun-associated redox partner CPRlun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450lun and CPRlun, we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14α-hydroxylation system to produce 14α-hydroxysteroids. Initial production of 14α-OH-AD (150 mg/L day productivity, 99% regioisomeric purity, and 60% w/w yield) and 14α-OH-RSS (64 mg/L day productivity, 40% regioisomeric purity, and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14α-hydroxysteroid production in the current yeast system.

Characterization of JEN family carboxylate transporters from the acid-tolerant yeast Pichia kudriavzevii and their applications in succinic acid production.

The unconventional yeast Pichia kudriavzevii is renowned for its ability to survive at low pH and has been exploited for the industrial production of various organic acids, especially succinic acid (SA). However, P. kudriavzevii can also utilize the di- and tricarboxylate intermediates of the Krebs cycle as the sole carbon sources for cell growth, which may adversely affect the extracellular accumulation of SA. Because the carboxylic acid transport machinery of P. kudriavzevii remains poorly understood, here, we focused on studying its SA transportation process from the perspective of mining and characterization of dicarboxylate transporters in a newly isolated acid-tolerant P. kudriavzevii strain CY902. Through genome sequencing and transcriptome analysis, two JEN family carboxylate transporters (PkJEN2-1 and PkJEN2-2) were found to be involved in SA transport. Substrate specificity analysis revealed that both PkJEN proteins are active dicarboxylate transporters, that can effectively import succinate, fumarate and L-malate into the cell. In addition, PkJEN2-1 can transport α-ketoglutarate, while PkJEN2-2 cannot. Since PkJEN2-1 shows higher transcript abundance than PkJEN2-2, its role in dicarboxylate transport is more important than PkJEN2-2. In addition, PKJEN2-2 is also responsible for the uptake of citrate. To our best knowledge, this is the first study to show that a JEN2 subfamily transporter is involved in tricarboxylate transport in yeast. A combination of model-based structure analysis and rational mutagenesis further proved that amino acid residues 392-403 of the tenth transmembrane span (TMS-X) of PkJEN2-2 play an important role in determining the specificity of the tricarboxylate substrate. Moreover, these two PkJEN transporters only exhibited inward transport activity for SA, and simultaneous inactivation of both PkJEN transporters reduced the SA influx, resulting in enhanced extracellular accumulation of SA in the late stage of fermentation. This work provides useful information on the mechanism of di-/tricarboxylic acid utilization in P. kudriavzevii, which will help improve the organic acid production performance of this microbial chassis.