Construction of a cellulose-metabolizing Komagataella phaffii (Pichia pastoris) by co-expressing glucanases and ß-glucosidase.

Cellulose is a highly available and renewable carbon source in nature. However, it cannot be directly metabolized by most microbes including Komagataella phaffii (formerly Pichia pastoris), which is a frequently employed host for heterologous protein expression and production of high-value compounds. A K. phaffii strain was engineered that constitutively co-expresses an endoglucanase and a ß-glucosidase both from Aspergillus niger and an exoglucanase from Trichoderma reesei under the control of bidirectional promoters. This engineered strain was able to grow on cellobiose and carboxymethyl cellulose (CMC) but not on Avicel. However, the detected release of cellobiose from Avicel by using the produced mixture of endoglucanase and exoglucanase as well as the released glucose from Avicel by using the produced mixture of all three cellulases at 50 °C indicated the production of exoglucanase under the liquid culture conditions. The successful expression of three cellulases in K. phaffii demonstrated the feasibility to enable K. phaffii to directly use cellulose as a carbon source for producing recombinant proteins or other high-value compounds.

Lifespan extension by methionine restriction requires autophagy-dependent vacuolar acidification.

Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization.

Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature's use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, ß-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic hosts, enabling a myriad of metabolic engineering and synthetic biology applications.

Publisher Correction: Engineered bidirectional promoters enable rapid multi-gene co-expression optimization.

The original version of this Article was updated after publication to add the ORCID ID of the author Thomas Vogl, which was inadvertently omitted, and to include a corrected version of the 'Description of Additional Supplementary Files' which originally lacked legends for each file.

A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris.

The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the alcohol oxidase 1 gene (PAOX1). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the pentose phosphate pathway (PPP) and genes involved in the defense of reactive oxygen species (ROS), providing strong promoters that, in part, even outperform PAOX1 and offer novel regulatory profiles. We have applied these tightly regulated promoters together with novel terminators as useful tools for the expression of a heterologous biosynthetic pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host.

Synthetic core promoters for Pichia pastoris.

Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as Saccharomyces cerevisiae and Pichia pastoris have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for P. pastoris, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural AOX1 promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields.

Autophagy extends lifespan via vacuolar acidification.

Methionine restriction (MetR) is one of the rare regimes that prolongs lifespan across species barriers. Using a yeast model, we recently demonstrated that this lifespan extension is promoted by autophagy, which in turn requires vacuolar acidification. Our study is the first to place autophagy as one of the major players required for MetR-mediated longevity. In addition, our work identifies vacuolar acidification as a key downstream element of autophagy induction under MetR, and possibly after rapamycin treatment. Unlike other amino acids, methionine plays pleiotropic roles in many metabolism-relevant pathways. For instance, methionine (i) is the N-terminal amino acid of every newly translated protein; (ii) acts as the central donor of methyl groups through S-adenosyl methionine (SAM) during methylation reactions of proteins, DNA or RNA; and (iii) provides the sulfhydryl groups for FeS-cluster formation and redox detoxification via transsulfuration to cysteine. Intriguingly, MetR causes lifespan extension, both in yeast and in rodents. We could show that in Saccharomyces cerevisiae, chronological lifespan (CLS) is increased in two specific methionine-auxotrophic strains (namely Δmet2 and Δmet15).